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PubMed1985

Conformational changes in Mycobacterium smegmatis glutamine synthetase induced by certain divalent cations.

Manganese ion, like Mg2+, has been found to produce high biosynthetic activity of the unadenylylated form of glutamine synthetase obtained from Mycobacterium smegmatis, and the activity with each of these cations was decreased by the adenylylation of the enzyme. Further, the gamma-glutamyltransferase reaction was catalyzed in the presence of either Mn2+, Mg2+, or Co2+ with both unadenylylated and adenylylated enzyme; however, each of these divalent cation-dependent activities was also decreased by one order of magnitude by adenylylation of the enzyme. From studies of UV-difference spectra, it was found that the ability of M. smegmatis glutamine synthetase to assume a number of distinctly different configurations was the result of the varied response of the enzyme to different cations. When either Mn2+, Mg2+, Ca2+, or Co2+ was added to the relaxed (divalent cation-free) enzyme at saturated concentration, each produced a similar UV-difference spectrum of the enzyme, indicating that the conformational states induced by these cations are similar with respect to the polarity of the microenvironment surrounding the tyrosyl and tryptophanyl groups of the enzyme. The binding of Cd2+, Ni2+, or Zn2+ to the relaxed enzyme each produced a different shift in the UV-absorption spectrum of the enzyme, indicating different conformational states. The kinetics of the spectral change that occurred upon addition of Mn2+, Mg2+, or Co2+ to a relaxed enzyme preparation were determined. The first-order rate constants for the decrease in relaxed enzyme with Mn2+ and Mg2+ were 0.604 min-1 and 0.399 min-1, respectively, at 25 degrees C, pH 7.4. The spectral change with Co2+ was completed within the time of mixing (less than 4 s). For these three metal ions, the total spectral change as well as the time course of the change were the same for both the unadenylylated enzyme and the partially adenylylated enzyme. However, Hill coefficients obtained from spectrophotometric titration data for both Mn2+ and Mg2+ were decreased with adenylylated enzyme to compared with unadenylylated enzyme. These results suggest that covalently bound AMP on each subunit may be involved in subunit interactions within the dodecamer. Circular dichroism measurements also indicated that the various structural changes of the M. smegmatis glutamine synthetase were produced by the binding of the divalent cations.

Adenosine Monophosphate↗
PubMed1987

Cationic modulation of follicle-stimulating hormone binding to granulosa cell receptor.

Magnesium (Mg2+) increases binding of follicle-stimulating hormone (FSH) to membrane-bound receptors and increases adenylyl cyclase activity. We examined the effects of divalent and monovalent cations on FSH binding to receptors in granulosa cells from immature porcine follicles. Divalent and monovalent cations increased binding of [125I]iodo-porcine FSH (125I-pFSH). The divalent cations Mg2+, calcium (Ca2+) and manganese, (Mn2+) increased specific binding a maximum of 4- to 5-fold at added concentrations of 10 mM. Mg2+ caused a half-maximal enhancement of binding at 0.6 mM, whereas Ca2+ and Mn2+ had half-maximal effects at 0.7 mM and 0.8 mM, respectively. The monovalent cation potassium (K+) increased binding a maximum of 1.5-fold at an added concentration of 50 mM, whereas the monovalent cation (Na+) did not increase binding at any concentration tested. The difference between K+ and Na+ suggested that either enhancement of binding was not a simple ionic effect or Na+ has a negative effect that suppresses its positive effect. Ethylenediamine tetraacetic acid, a chelator of Mg2+, prevented binding of 125I-pFSH only in the presence of Mg2+, whereas pregnant mare's serum gonadotropin, a competitor with FSH for the receptor, prevented binding in both the absence and the presence of Mg2+. Guanyl-5-ylimidodiphosphate (Gpp[NH]p) inhibited binding of 125I-pFSH in the absence or presence of Mg2+, but only at Gpp(NH)p concentrations greater than 1 mM. We used Mg2+ to determine if divalent cations enhanced FSH binding by increasing receptor affinity or by increasing the apparent number of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Neutrophil cytotoxicity induced by immune complexes prepared with cationized antibodies.

Here we analyse the ability of soluble immune complexes (IC) prepared with cationized antibodies to induce cytotoxic responses mediated by neutrophils. While cationized IC induced high levels of cytotoxicity, control IC induced very low levels of response. Inhibition of cytotoxicity by catalase but not by three haemenzyme inhibitors suggests that oxygen-dependent but myeloperoxidase-independent mechanisms are responsible for cytolysis. While the response induced by control IC was enhanced by cytochalasin B and was not modified by colchicine, that induced by cationized IC was markedly inhibited by cytochalasin B and significantly enhanced by colchicine. Cytotoxicity induced by cationized IC was completely abrogated by monoclonal antibodies to Fc gamma RII. Using control IC, a partial inhibition was observed employing either anti-Fc gamma RII or anti-Fc gamma RIII monoclonal antibodies. Treatment of neutrophils with chemotrypsin or pronase significantly enhanced cytotoxicity induced by cationized IC but not by control IC. We also found that non-specific absorptive mechanisms appear to play an important role in the binding of cationized IC, but not control IC, to the neutrophil surface. The significance of these results is discussed.

Antibodies↗
PubMed1977

Dependence of tRNA structure in solution upon ionic condition of the solvent. Fluorescence studies of monovalent cation binding to tRNAPhe from barley embryos.

Dependence of barley phenylalanine tRNA (tRNAPhe) fluorescence intensity at 430 nm upon LiCl, NaCl, KCl, CsCl or NH4Cl concentration was measured in 0.01 M Tris-HCl, pH 7.5, 0.001 M Na2EDTA solutions. Increase of monovalent cation concentration in the solvent from 0 to 2 M induced about 3-fold fluorescence intensity enhancement. The fractional fluorescence change was used as a measure of bound ligand concentration. Fluorescence Scatchard plots revealed three classes of monovalent cation binding sites on the tRNA molecule: interacting (strong) and independent (weak and very weak) sites. Calculated from Scatchard plots binding constants (K), for strong and weak binding of monovalent cations (in the case of Na+ binding: Ks = 26 M-1 and Kw = 4.3 M-1 respectively) exhibit linear dependence upon ionic radius (r). Two limiting values obtained from the plot of K versus r: K(max) at r = 0 r(max) at K = 0, characterize additionally strong and weak monovalent cation binding sites (Ks(max) = 42 M-1 Kw(max) = 8.5 M-1, rs(max) = 0.23 nm and rw(max) = 0.22 nm). A model of the relationship between weak Mg2+ binding sites and monovalent cation binding sites as well as of monovalent cations binding to tRNA is proposed.

Cations, Monovalent↗
PubMed2000

The heterodimeric amino acid transporter 4F2hc/LAT1 is associated in Xenopus oocytes with a non-selective cation channel that is regulated by the serine/threonine kinase sgk-1.

System L is the major Na(+)-independent amino acid transporter of mammalian cells. It is constituted of the type II membrane protein 4F2hc (CD98) which is covalently linked to the polytopic membrane protein LAT1 via a disulfide bridge. The transporter is known to be regulated by the mineral corticoid aldosterone in Xenopus A6 cells. To understand the regulation of the transporter, the 4F2hc/LAT1 heterodimer was functionally expressed in Xenopus laevis oocytes and its transport properties were analysed using flux measurements and the two-electrode voltage-clamp technique. Expression of 4F2hc/LAT1 resulted in a rapid increase in a Na(+)-independent neutral amino acid antiport activity and simultaneously gave rise to a cation conductance. The cation channel was non-rectifying and non-selective, conducting Li(+) > Cs(+) = Na(+) > K(+). After replacement of Na(+) by NMDG, however, the currents were suppressed almost completely. The cation channel was not inhibited by amiloride, Ba2(+), TEA, Hoe293B, flufenamic acid or substrates of the system L amino acid transporter. Significant inhibition, however, was observed in the presence of La3(+), Gd3(+) and quinidine. Channel activity was upregulated by coexpression of 4F2hc/LAT1 with the aldosterone-regulated protein kinase sgk-1. The cation conductance was sensitive to changes in the redox potential, being inhibited following incubation of the oocytes with DTE for 30 min. Mutation of either of the disulfide bridge-constituting cysteines to serine resulted in a loss of ion channel activity whereas amino acid transport was unaffected. It is concluded that the 4F2hc/LAT1 heterodimer regulates a closely associated cation channel or even constitutes a cation channel itself.

Amino Acid Transport Systems↗
PubMed1990

Quinone sensitized electron transfer photooxidation of nucleic acids: chemistry of thymine and thymidine radical cations in aqueous solution.

The 2-methyl-1,4-naphthoquinone (MQ) sensitized photooxidation of nucleic acid derivatives has been studied by laser flash photolysis and steady state methods. Thymine and thymidine, as well as other DNA model compounds, quench triplet MQ by electron transfer to give MQ radical anions and pyrimidine or purine radical cations. Although the pyrimidine radical cations cannot be directly observed by flash photolysis, the addition of N,N,N',N'-tetramethyl-1,4-phenylenediamine (TMPD) results in the formation of the TMPD radical cation via scavenging of the pyrimidine radical cation. The photooxidation products for thymine and thymidine are shown to result from subsequent chemical reactions of the radical cations in oxygenated aqueous solution. The quantum yield for substrate loss at limiting substrate concentrations is 0.38 for thymine and 0.66 for thymidine. The chemistry of the radical cations involves hydration by water leading to C(6)-OH adduct radicals of the pyrimidine and deprotonation from the N(1) position in thymine and the C(5) methyl group for thymidine. Superoxide ions produced via quenching of the quinone radical anion with oxygen appear to be involved in the formation of thymine and thymidine hydroperoxides and in the reaction with N(1)-thyminyl radicals to regenerate thymine. The effects of pH were examined in the range pH 5-8 in both the presence and absence of superoxide dismutase. Initial C(6)-OH thymine adducts are suggested to dehydrate to give N(1)-thyminyl radicals.

Cations↗
PubMed2001

Interaction between store-operated non-selective cation channels and the Na(+)-Ca(2+) exchanger during secretion in the rat colon.

The properties of capacitative Ca(2+) influx were studied using the whole-cell patch-clamp technique in crypts isolated from rat distal colon. Store-operated cation influx was evoked by increasing the intracellular buffering capacity for Ca(2+) in the pipette solution; contamination by Cl(-) currents was reduced by the use of NMDG gluconate as the main electrolyte in the pipette solution. The permeability of the non-selective cation conductance stimulated by store depletion had the following sequence for monovalent cations: Cs(+) > Na(+) > or = Li(+). The store-operated conductance is permeable to Na(+) and Ca(2+), but in contrast to Na(+), Ca(2+) also exerts a (feedback) inhibition on its own influx. Other divalent cations shared this inhibitory action with the sequence: Ca(2+) > or = Mg(2+) > or = Ba(2+) > or = Sr(2+). Fura-2 experiments revealed that replacement of extracellular Na(+) by NMDG(+) induced an increase in the intracellular Ca(2+) concentration, which was suppressed by the Na(+)-Ca(2+) exchange inhibitor, dichlorobenzamil, indicating the presence of a Na(+)-Ca(2+) exchanger within the colonic crypt cells. In Ussing chamber experiments dichlorobenzamil induced an increase in short-circuit current (I(sc)) in the majority of tissues tested indicating that this exchanger acts as a Ca(2+)-extruding transporter under physiological conditions. When Ca(2+)-dependent anion secretion was stimulated by the acetylcholine analogue carbachol, dichlorobenzamil no longer evoked an increase in I(sc), indicating that after stimulation of the store-operated cation conductance the Na(+)-Ca(2+) exchanger is turned off. Therefore, it is concluded that the influx of Na(+) across the non-selective store-operated cation conductance serves to reduce the driving force for Ca(2+) extrusion via the Na(+)-Ca(2+) exchanger and thereby maintains the increase in the intracellular Ca(2+) concentration during induction of secretion. Experimental Physiology (2001) 86.4, 461-468.

Amiloride↗
PubMed1988

Activation and blockage of a calcium-sensitive cation-selective pathway in the apical membrane of toad urinary bladder.

1. The properties of cation movements through a previously described Ca2+-sensitive oxytocin-stimulated pathway in the apical membrane of the toad urinary bladder were further investigated. 2. In the absence of Ca2+ and other polyvalent cations in the mucosal medium, oxytocin markedly stimulated the flow of current from mucosa to serosa when the major cation in the mucosal solution was any of the following ions: Na+, K+, Rb+, Cs+ or Li+. Analysis of the current noise showed a Lorentzian component associated with the movement of these cations. 3. Ca2+ and other divalent cations in the mucosal solution depressed both the current and the Lorentzian component of the fluctuation spectra. The Michaelis-Menten constants were 2.5, 10 and 58 mumol/l for Ca2+, Sr2+ and Mg2+ respectively. 4. The dihydropyridine Ca2+ channel blockers nitrendipine (10(-5) mol/l) and nicardipine (10(-6) mol/l) inhibited the Ca2+-sensitive current. 5. Alterations of the mucosal pH showed that the current and the plateau of the Lorentzian component increased by elevating the pH from 6 to 8. The Ca2+-sensitive current was further stimulated by increasing pH to 9. However, this manoeuvre resulted in the disappearance of the Lorentzian component in the noise spectrum. 6. Increasing either mucosal [Na+] or [K+] up to 115 mmol/l did not lead to saturation of the current passing through the Ca2+-sensitive channel. In contrast the amiloride-sensitive channel showed saturating behaviour when mucosal [Na+] was increased; half-maximum current was reached when mucosal [Na+] was about 15 mmol/l. 7. When a Na+-free mucosal solution, prepared with either choline or TEA as major cation, was rapidly replaced by a solution with Na+ (115 mmol/l), the current through the Ca2+-sensitive channel increased rapidly and then remained at a nearly constant level. This behaviour is in contrast with the response of the current through the amiloride-sensitive pathway. After suddenly increasing mucosal Na+ concentration ([Na+]m), the current through this channel first increased rapidly and then declined to values of nearly 50% of the peak about 10 min after the increase in [Na+]m.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Properties of a constitutively active Ca2+-permeable non-selective cation channel in rabbit ear artery myocytes.

In smooth muscle, non-selective cation conductances contribute to agonist-evoked depolarisation and contraction, and in the present study using patch-pipette techniques we describe the properties of a constitutively active cation channel. With whole-cell recording in K+-free conditions, there was a spontaneous current with a reversal potential (Er) that was altered by replacement of external Na+ by an impermeant cation, but not when external Cl- was replaced by an impermeant anion. The tonic cation inward current could be carried by Ca2+ ions and was greatly enhanced when the external Ca2+ concentration was reduced. In outside-out patches there was spontaneous cation channel activity that could be resolved into three conductance states of about 15, 25 and 40 pS, all with the same Er as the whole-cell current. Kinetic analysis revealed that there were two open times of about 1 and 5 ms and that the currents displayed bursting kinetics with burst durations of approximately 5 ms and 25 ms. Removal of external Ca2+ ions increased the probability of channel opening (Po) sixfold, which was associated with an increase in the longer burst duration. Bath application of the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol increased Po, but phorbol 12,13-dibutyrate, which stimulates protein kinase C (PKC), reduced channel activity. In contrast, the PKC inhibitor chelerythrine increased the activity of channel currents. It is concluded that in rabbit ear artery myocytes there is a constitutively active Ca2+-permeable cation channel that is regulated by external Ca2+ ions and suppressed by tonic PKC activity. It is proposed that this mechanism may contribute to the resting membrane conductance and basal Ca2+ influx in this particular arterial preparation.

Alkaloids↗
PubMed2004

The N-methyl-D-aspartate receptor inhibitory potencies of aromatic inhaled drugs of abuse: evidence for modulation by cation-pi interactions.

Benzene and several close structural analogs are inhaled drugs of abuse with general anesthetic activity. By virtue of their pi electron clouds, they may engage in attractive electrostatic interactions with cationic atomic charges on protein targets. In this study, we tested the hypothesis that inhaled drugs of abuse inhibit human N-methyl-D-aspartate (NMDA) receptors with potencies that correlate with their abilities to engage in cation-pi interactions. Electrophysiological techniques were used to define the NR1/NR2B NMDA receptor inhibitory concentrations of volatile benzene analogs, and computer modeling was used to quantify their abilities to engage in cation-pi interactions and their molecular volumes. In addition, each compound's octanol/gas partition coefficient (a measure of hydrophobicity) was quantified. All 18 compounds inhibited human NR1/NR2B NMDA receptors reversibly and in a concentration-dependent manner. NMDA receptor inhibitory potency correlated strongly with the ability to engage in cation-pi interactions, weakly with hydrophobicity, and was independent of molecular volume. This is consistent with the hypothesis that cation-pi interactions enhance the binding of inhaled drugs of abuse to the NMDA receptor and suggests that the receptor binding site(s) for these drugs possesses significant cationic character.

Animals↗
PubMed1992

Down-regulation of cystic fibrosis transmembrane conductance regulator gene expression by agents that modulate intracellular divalent cations.

In cystic fibrosis (CF), epithelial cells are unable to normally up-regulate apical membrane Cl- secretion in response to agents which increase cyclic AMP, but they do increase Cl- secretion in response to increases in intracellular Ca2+. Since intracellular divalent cations regulate the expression of many genes, we hypothesized that mobilization of intracellular Ca2+ and/or other divalent cations might modulate not only Ca(2+)-dependent Cl- channels but also cystic fibrosis transmembrane conductance regulator (CFTR) gene expression. To evaluate this concept, HT-29 human colon carcinoma cells were cultured under various conditions designed to manipulate intracellular divalent cation concentrations and CFTR gene expression was quantified at the levels of transcription, mRNA accumulation, mRNA half-life, and protein. Exposure to the divalent cation ionophores A23187 and ionomycin (agents which increase intracellular divalent cation concentrations) caused dose- and time-dependent reductions of CFTR mRNA levels, which could be blocked by the use of Ca(2+)- and Mg(2+)-free media. Ionophore-induced CFTR gene modulation was also observed with T84 human colon carcinoma cells and freshly isolated normal human bronchial epithelial cells. Incubation of HT-29 cells with thapsigargin, an agent that releases Ca2+ from intracellular stores, or in medium containing increased extracellular concentrations of Ca2+ or Mg2+ also caused down-regulation of CFTR mRNA levels. Transcription run-on analysis showed that, parallel with the decrease in CFTR mRNA levels, A23187 reduced the rate of transcription of the CFTR gene, while CFTR mRNA transcript half-life was unaffected. Consistent with the down-regulation of CFTR gene expression, CFTR protein levels also decreased after exposure to A23187. Thus, despite the independence of Ca(2+)-dependent Cl- channels and cyclic AMP-dependent CFTR-related Cl- channels in epithelial cells, increases in intracellular divalent cation concentrations down-regulate the expression of the CFTR gene at the transcriptional level, with consequent decreases in CFTR mRNA and protein.

Calcimycin↗
PubMed1991

Role of cations in the flocculation of Saccharomyces cerevisiae and discrimination of the corresponding proteins.

Asexual yeast flocculation was studied using strong flocculents of Saccharomyces cerevisiae. The inhibitory effect of cations on flocculation is considered to be caused by competition between those cations and Ca2+ at the binding site of the Ca(2+)-requiring protein that is involved in flocculation. Inhibition of flocculation by various cations occurred in the following order: La3+, Sr2+, Ba2+, Mn2+, Al3+, and Na+. Cations such as Mg2+, Co2+, and K+ promoted flocculation. This promoting effect may be based on the reduction of electrostatic repulsive force between cells caused by binding of these cations anionic groups present on the cell surface. In flocculation induced by these cations, trace amounts of Ca2+ excreted on the cell surface may activate the corresponding protein. The ratio of Sr2+/Ca2+ below which cells flocculated varied among strains: for strains having the FLO5 gene, it was 400 to 500; for strains having the FLO1 gene, about 150; and for two alcohol yeast strains, 40 to 50. This suggests that there are several different types of cell surface proteins involved in flocculation in different yeast strains.

Adsorption↗
PubMed2006

TRPM7 is a stretch- and swelling-activated cation channel involved in volume regulation in human epithelial cells.

Stretch- and swelling-activated cation (SSAC) channels play essential roles not only in sensing and transducing external mechanical stresses but also in regulating cell volume in living cells. However, the molecular nature of the SSAC channel has not been clarified. In human epithelial HeLa cells, single-channel recordings in cell-attached and inside-out patches revealed expression of a Mg(2+)- and Gd(3+)-sensitive nonselective cation channel that is exquisitely sensitive to membrane stretch. Whole cell recordings revealed that the macroscopic cationic currents exhibit transient receptor potential (TRP) melastatin (TRPM)7-like properties such as outward rectification and sensitivity to Mg(2+) and Gd(3+). The whole cell cation current was augmented by osmotic cell swelling. RT-PCR and Western blotting demonstrated molecular expression of TRPM7 in HeLa cells. Treatment with small interfering RNA (siRNA) targeted against TRPM7 led to abolition of single stretch-activated cation channel currents and of swelling-activated, whole cell cation currents in HeLa cells. The silencing of TRPM7 by siRNA reduced the rate of cell volume recovery after osmotic swelling. A similar inhibition of regulatory volume decrease was also observed when extracellular Ca(2+) was removed or Gd(3+) was applied. It is thus concluded that TRPM7 represents the SSAC channel endogenously expressed in HeLa cells and that, by serving as a swelling-induced Ca(2+) influx pathway, it plays an important role in cell volume regulation.

Cell Line↗
PubMed1981

Effects of divalent cations on beta-cell electrical activity.

The effects of various divalent cations, added to the external medium, upon beta-cell action potential were studied using intracellular microelectrodes. Changes of spike peak potential, as a function of external cation concentration, indicate that Sr2+ or Ba2+ may substitute for Ca2+ as a charge carrier. Complete blockage by Mn2+ of electrical activity elicited by Sr2+, Ba2+, or Ca2+ suggests that these cations penetrate the membrane though the same Ca2+ channel. The increase of maximum rate of depolarization, dV(d)/dtmax, and decrease of maximum rate of repolarization, dV(r)/dtmax, when Sr2+ is substituted for Ca2+ suggest that Sr2+ penetrates more readily the Ca2+ channel but is less effective than Ca2+ in activating K permeability. Reversal of these effects by addition of equimolar Ca2+ to Sr2+ indicates that Ca2+ has a greater affinity than Sr2+ for the receptor site. The blockage of electrical activity by Ba2+ at a depolarized membrane level suggests that Ba2+ markedly reduces all K+ permeabilities. Analysis of dV(d)/dtmax at various Ca2+ concentrations, in the presence of nonpermeant divalent cations (Co2+, Mn2+, and Mg2+), shown that these cations bind competitively at the same receptor site with differing dissociation constants, For all of these divalent cations, the order of binding would be Co2+ greater than Mn2+ greater than Ca2+ greater than Sr2+, Mg2+.

Action Potentials↗
PubMed1995

Optimization of cationic lipid-mediated gene transfer to airway epithelia.

The use of cationic lipids for gene transfer to airway epithelia has shown promise in in vitro and in vivo studies. However, previous studies have used a wide variety of different lipid preparations and different formulations. Few studies have been designed to optimize the variables involved in transfection, and none have been focused on airway epithelia. Therefore we examined variables that affect cationic lipid-mediated transfection of HeLa cells and of airway epithelial cells grown on permeable filter supports at the air-liquid interface. To quantitate expression of cDNA, we assayed expression of luciferase. We found that the ratio of DNA to lipid was an important variable that determined transfection efficiency. In both HeLa and airway epithelial cells, the optimum charge ratio of cationic lipid to anionic DNA was approximately 1.25, consistent with the notion that a positively charged complex facilitates interaction with the negatively charged cell membrane. After testing a series of readily available cationic lipids, we found that 1,2-dimyristyloxypropyl-N,N-dimethyl-hydroxyethyl ammonium bromide (DMRIE)/dioleoyl phosphatidylethandamine (DOPE) appeared to show good efficacy. The concentration of DNA and cationic lipid also played an important role: in HeLa cells the optimum concentration of cationic lipid was approximately 5 microM and in airway epithelial cells was approximately 15 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Cations↗
PubMed1991

Organic cation transport in human renal brush-border membrane vesicles.

The renal proximal tubule is responsible for the active elimination of organic cations. Studies in brush-border membrane vesicles (BBMV) suggest that active organic cation transport is mediated by an organic cation-proton antiporter. The goals of this study were to determine whether this transporter is expressed in human kidney and to elucidate its characteristics. Transport of the organic cations N1-[3H]methylnicotinamide (NMN) and [14C]tetraethylammonium (TEA) was determined by rapid filtration in BBMV of donor human kidneys. The uptake of NMN and TEA was driven against a concentration gradient by an outwardly directed proton gradient. NMN uptake was inhibited by the organic cations TEA, NMN, quinine, and cimetidine, but was not affected by p-aminohippuric acid, cephalexin, and polyamines. The electroneutral transport of NMN was characterized by a Km of 0.44 +/- 0.07 mM and a Vmax of 24.4 +/- 15.2 pmol.mg protein-1.s-1. The rate of proton efflux from BBMV increased in the presence of an inwardly directed TEA gradient. Preloading BBMV with NMN or TEA trans-stimulated the initial rate of uptake of TEA. Therefore the human proximal tubule expresses an organic cation-proton antiporter in the brush-border membrane. The antiporter is similar to that described in other mammalian species but shows some differences in its transport characteristics.

Adult↗
PubMed1991

Inhibition of 5-HT3 receptor-mediated ion current by divalent metal cations in NCB-20 neuroblastoma cells.

1. The effect of micromolar concentrations of divalent metal cations on ion current activated by 5-hydroxytryptamine (5-HT) was investigated in NCB-20 neuroblastoma cells by the use of the whole-cell, patch-clamp technique. 2. Ion current activated by 5-HT in these cells was mimicked by 5-HT3 receptor agonists, blocked by nanomolar concentrations of selective 5-HT3 receptor antagonists and reversed polarity at approximately 0 mV. These properties indicate that this current is carried primarily if not exclusively by the nonspecific cation channel activated by the 5-HT3 receptor. 3. The Group IIb metal cations Cd2+ and Zn2+ and the Group Ib cation Cu2+ inhibited 5-HT-activated current with inhibition increasing in a concentration-dependent manner over micromolar concentrations of the ions. The order of potency of the ions for inhibiting 5-HT-activated current was Zn2+ (IC50 = 20 microM) greater than or equal to Cu2+ (IC50 = 25 microM) greater than Cd2+ (IC50 = 75 microM) at -50 mV. The other divalent metal cations tested (Ba2+, Co2+, Mg2+, Mn2+, and Ni2+) produced little or no inhibition of 5-HT-activated current at concentrations up to 200 microM. 4. Inhibition of 5-HT-activated current by Cd2+ and Zn2+ was dependent on membrane potential with the Kd increasing e-fold per 72 and 52 mV, respectively. Inhibition by Cu2+ was much less voltage dependent with the Kd increasing e-fold per 233 mV. 5. Inhibition by all three cations decreased with increasing concentration of agonist over a range of 5-HT concentrations from 1 to 10 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Effects of anions on ATP-activated nonselective cation current in NG108-15 cells.

Extracellular ATP induces a nonselective cation current and elevates intracellular Ca2+ concentration via P2Z receptors in NG108-15 cells. We found that the ATP-induced nonselective cation current became larger in methanesulfonic acid (MS-) than in Cl- external solution. We therefore examined the effects of various external anions on the ATP-induced cation current with the use of the whole cell patch-clamp technique. The concentration-response curves for ATP were obtained in different anionic external solutions. The maximum current density (Imax) and the concentration of agonist that gives 50% of maximum response (EC50) value of ATP were obtained by fitting the curves with the use of the Hill coefficient of 2. The apparent Imax decreased in the order of aspartic acid (Asp-) > MS- > F- > Cl- > Br- > or = I-. The apparent EC50 values for ATP were shifted to the right in the sequence of Asp- < F- < MS- < Br- < Cl- < I-. Thus both Imax and EC50 values were affected by anions, indicating that anions are mixed-type inhibitors of the ATP-induced current. The shift of the EC50 values of ATP indicates that anions interfere with ATP binding to the receptor. External Cl- was a noncompetitive inhibitor with respect to external Na+, a major cation carrying the ATP-induced current. We conclude that extracellular anions inhibit the ATP-induced nonselective cation current at least partly by interfering with ATP binding to the P2Z receptor, which is associated with the nonselective cation channels.

Acetylcholine↗