Inhibition of anion permeability by amphiphilic compounds in human red cell: Evidence for an interaction of niflumic acid with the band 3 protein

Summary In human erythrocyte, permeability to the anion is instantaneously, reversibly, and noncompetitively inhibited by the nonsteroidal anti-inflammatory drug, niflumic acid. The active form of this powerful inhibitor (I ₅₀=6×10^–7 m) is the ionic form. We demonstrated that: (i) The binding of niflumic acid to the membrane of unsealed ghosts shows one saturable and one linear component over the concentration range studied. The saturable component vanishes when chloride transport is fully inhibited by covalently bound 4-acetamido-4-isothiocyano stilbene-2,2-disulfonic acid (SITS). Our estimate of these SITS protectable niflumate binding sites (about 9×10⁵ per cell) agrees with the number of protein molecules per cell in band 3. These sites are halfsaturated with 10^–6 m niflumic acid, a concentration very close toI ₅₀. (ii) Niflumic acid inhibits the binding reaction of SITS with anion controlling transport sites. These results indicate that niflumic acid and SITS are mutually exclusive inhibitors, suggesting that niflumic acid interacts with the protein in band 3.Niflumic acid also decreases glucose and ouabain-insensitive sodium permeabilities. However, these effects are produced at a very high concentration of niflumic acid (in millimolar range), suggesting unspecific action, possibly through lipid phase.     

Activation of Slo2.1 channels by niflumic acid

Slo2.1 channels conduct an outwardly rectifying K⁺ current when activated by high [Na⁺]_i. Here, we show that gating of these channels can also be activated by fenamates such as niflumic acid (NFA), even in the absence of intracellular Na⁺. In Xenopus oocytes injected with <10 ng cRNA, heterologously expressed human Slo2.1 current was negligible, but rapidly activated by extracellular application of NFA (EC₅₀ = 2.1 mM) or flufenamic acid (EC₅₀ = 1.4 mM). Slo2.1 channels activated by 1 mM NFA exhibited weak voltage dependence. In high [K⁺]_e, the conductance–voltage (G-V) relationship had a V_1/2 of +95 mV and an effective valence, z, of 0.48 e. Higher concentrations of NFA shifted V_1/2 to more negative potentials (EC₅₀ = 2.1 mM) and increased the minimum value of G/G_max (EC₅₀ = 2.4 mM); at 6 mM NFA, Slo2.1 channel activation was voltage independent. In contrast, V_1/2 of the G-V relationship was shifted to more positive potentials when [K⁺]_e was elevated from 1 to 300 mM (EC₅₀ = 21.2 mM). The slope conductance measured at the reversal potential exhibited the same [K⁺]_e dependency (EC₅₀ = 23.5 mM). Conductance was also [Na⁺]_e dependent. Outward currents were reduced when Na⁺ was replaced with choline or mannitol, but unaffected by substitution with Rb⁺ or Li⁺. Neutralization of charged residues in the S1–S4 domains did not appreciably alter the voltage dependence of Slo2.1 activation. Thus, the weak voltage dependence of Slo2.1 channel activation is independent of charged residues in the S1–S4 segments. In contrast, mutation of R190 located in the adjacent S4–S5 linker to a neutral (Ala or Gln) or acidic (Glu) residue induced constitutive channel activity that was reduced by high [K⁺]_e. Collectively, these findings indicate that Slo2.1 channel gating is modulated by [K⁺]_e and [Na⁺]_e, and that NFA uncouples channel activation from its modulation by transmembrane voltage and intracellular Na⁺.     

Inhibition ofListeria monocytogenes by plantaricin NA, an antibacterial substance fromLactobacillus plantarum

ALactobacillus plantarum of vegetable origin produced a bacteriocin inhibitory toListeria monocytogenes. The antimicrobial agent was inactivated by proteolytic enzymes, was resistant to heat (100°C for 30 min) and stable over a wide pH range (pH 2–10), and displayed a bactericidal mode of action. Growth inhibition ofL. monocytogenes depend on bacteriocin concentration. The antilisterial efficiency depended on the strain ofL. monocytogenes used but was not influenced by the growth phase of this strain. A decrease in absorbance overtime, indicative of cell lysis, was also observed. The significance of the results is discussed in relation to the potential of the bacteriocin in controllingListeria-associated food-borne hazards in foods.     

Inhibition of the cardiac inward rectifier potassium currents by KB-R7943

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea) was developed as a specific inhibitor of the sarcolemmal sodium–calcium exchanger (NCX) with potential experimental and therapeutic use. However, KB-R7943 is shown to be a potent blocker of several ion currents including inward and delayed rectifier K⁺ currents of cardiomyocytes. To further characterize KB-R7943 as a blocker of the cardiac inward rectifiers we compared KB-R7943 sensitivity of the background inward rectifier (I_K1) and the carbacholine-induced inward rectifier (I_KACh) currents in mammalian (Rattus norvegicus; rat) and fish (Carassius carassius; crucian carp) cardiac myocytes. The basal I_K1 of ventricular myocytes was blocked with apparent IC50-values of 4.6 × 10^− 6 M and 3.5 × 10^− 6 M for rat and fish, respectively. I_KACh was almost an order of magnitude more sensitive to KB-R7943 than I_K1 with IC₅₀-values of 6.2 × 10^− 7 M for rat and 2.5 × 10^− 7 M for fish. The fish cardiac NCX current was half-maximally blocked at the concentration of 1.9–3 × 10^− 6 M in both forward and reversed mode of operation. Thus, the sensitivity of three cardiac currents to KB-R7943 block increases in the order I_K1 ~ I_NCX < I_KACh. Therefore, the ability of KB-R7943 to block inward rectifier potassium currents, in particular I_KACh, should be taken into account when interpreting the data with this inhibitor from in vivo and in vitro experiments in both mammalian and fish models.     

Block of inward rectification by intracellular H+ in immature oocytes of the starfish Mediaster aequalis

Intracellular pH was recorded in immature starfish oocytes using pH- sensitive microelectrodes, and inwardly rectifying potassium currents were measured under voltage clamp. When the intracellular pH was lowered using acetate-buffered artificial sea water from the normal value of 7.09 to 5.9, inward rectification was completely blocked. The relationship between inward rectification and internal pH between 7.09 and 5.9 could be fit by a titration curve for the binding of three H ions to a site with a pK of 6.26 to block the channel. The H+ block showed no voltage dependence, and the activation kinetics of the inwardly rectifying currents were not affected by the changes in internal pH.     

The role of the cytoplasmic pore in inward rectification of Kir2.1 channels

Steeply voltage-dependent block by intracellular polyamines underlies the strong inward rectification properties of Kir2.1 and other Kir channels. Mutagenesis studies have identified several negatively charged pore-lining residues (D172, E224, and E299, in Kir2.1) in the inner cavity and cytoplasmic domain as determinants of the properties of spermine block. Recent crystallographic determination of the structure of the cytoplasmic domains of Kir2.1 identified additional negatively charged residues (D255 and D259) that influence inward rectification. In this study, we have characterized the kinetic and steady-state properties of spermine block in WT Kir2.1 and in mutations of the D255 residue (D255E, A, K, R). Despite minimal effects on steady-state blockade by spermine, D255 mutations have profound effects on the blocking kinetics, with D255A marginally, and D255R dramatically, slowing the rate of block. In addition, these mutations result in the appearance of a sustained current (in the presence of spermine) at depolarized voltages. These features are reproduced with a kinetic model consisting of a single open state, two sequentially linked blocked states, and a slow spermine permeation step, with residue D255 influencing the spermine affinity and rate of entry into the shallow blocked state. The data highlight a "long-pore" effect in Kir channels, and emphasize the importance of considering blocker permeation when assessing the effects of mutations on apparent blocker affinity.     

Structural basis of inward rectification: cytoplasmic pore of the G protein-gated inward rectifier GIRK1 at 1.8 A resolution

Inward rectifier K(+) channels govern the resting membrane voltage in many cells. Regulation of these ion channels via G protein-coupled receptor signaling underlies the control of heart rate and the actions of neurotransmitters in the central nervous system. We have determined the protein structure formed by the intracellular N- and C termini of the G protein-gated inward rectifier K(+) channel GIRK1 at 1.8 A resolution. A cytoplasmic pore, conserved among inward rectifier K(+) channels, extends the ion pathway to 60 A, nearly twice the length of a canonical transmembrane K(+) channel. The cytoplasmic pore is lined by acidic and hydrophobic amino acids, creating a favorable environment for polyamines, which block the pore. These results explain in structural and chemical terms the basis of inward rectification, and they also have implications for G protein regulation of GIRK channels.     

Inhibition of G-protein-coupled inward rectifying K+ channels by intracellular acidosis

G-protein-coupled inward rectification K(+) (GIRK) channels play an important role in modulation of synaptic transmission and cellular excitability. The GIRK channels are regulated by diverse intra- and extracellular signaling molecules. Previously, we have shown that GIRK1/GIRK4 channels are activated by extracellular protons. The channel activation depends on a histidine residue in the M1-H5 linker and may play a role in neurotransmission. Here, we show evidence that the heteromeric GIRK1/GIRK4 channels are inhibited by intracellular acidification. This inhibition was produced by selective decrease in the channel open probability with a modest drop in the single-channel conductance. The inhibition does not seem to require G-proteins as it was seen in two G-protein coupling-defective GIRK mutants and in excised patches in the absence of exogenous G-proteins. Three histidine residues in intracellular domains were critical for the inhibition. Individual mutation of His-64, His-228, or His-352 in GIRK4 abolished or greatly diminished the inhibition in homomeric GIRK4. Mutations of any of these histidine residues in GIRK4 or their counterparts in GIRK1 were sufficient to eliminate the pH(i) sensitivity of the heteromeric GIRK1/GIRK4 channels. Thus, the molecular and biophysical bases for the inhibition of GIRK channels by intracellular protons are illustrated. Because of the inequality of the pH(i) and pH(o) in most cells and their relatively independent controls by cellular versus systemic mechanisms, such pH(i) sensitivity may allow these channels to regulate cellular excitability in certain physiological and pathophysiological conditions when intracellular acidosis occurs.     

Molecular basis of inward rectification: polyamine interaction sites located by combined channel and ligand mutagenesis

Polyamines cause inward rectification of (Kir) K+ channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSEA+ modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter.     

Inhibition of lymphocyte mitogenesis by an arachidonic acid hydroperoxide

Incubation of murine spleen cells with the oxidation product of soybean lipoxidase-treated arachidonic acid results in profound inhibition of induction of proliferation and maturation of these cells. The active entity was shown to be the 15-hydroperoxide of arachidonic acid (15-HPAA). Inhibition of the enzymes of the cyclo-oxygenase pathway fails to disturb this effect, indicating that 15-HPAA is not a substrate for this series of enzymes. 15-HPAA produced in this manner interfered with RNA synthesis, DNA synthesis, and blastogenesis, while failing to exert cytotoxic effects on the cells themselves. A variety of lymphocyte subpopulations, distinguished by their responsiveness to a diverse group of mitogens, were all equally inhibited by the addition of 15-HPAA to culture. Addition of this agent even as late as 24 h after initiation of culture resulted in profound inhibition of the proliferative and differentiative responses of splenic B cells to bacterial lipopolysaccharide (LPS). Exposure of cells to 15-HPAA for 10–30 min was adequate to initiate inhibition, an event that exhibited marked temperature dependence. The effects of pre-incubation with 15-HPAA could not be reversed in its absence in recovery periods of up to 6 h prior to addition of LPS. The implications of these data with reference to cellular activation mechanisms are discussed.     

Delayed rectification in the cardiac Purkinje fiber is not activated by intracellular calcium

This study was designed to test the hypothesis that an outward current (Ix) responsible for action potential repolarization in the cardiac Purkinje fiber is activated by intracellular calcium (Cai). Pharmacological probes were combined with the measurement of membrane current and contractile activity under voltage clamp conditions. Experiments were designed to examine properties of Ix that have previously linked activation of this current to changes in Cai. The independence of Ix from Cai was demonstrated for each case tested. Thus, the results of these experiments support the view that Ix is not a calcium-activated current.     

Mg2+-dependent gating and strong inward rectification of the cation channel TRPV6

TRPV6 (CaT1/ECaC2), a highly Ca(2+)-selective member of the TRP superfamily of cation channels, becomes permeable to monovalent cations in the absence of extracellular divalent cations. The monovalent currents display characteristic voltage-dependent gating and almost absolute inward rectification. Here, we show that these two features are dependent on the voltage-dependent block/unblock of the channel by intracellular Mg(2+). Mg(2+) blocks the channel by binding to a site within the transmembrane electrical field where it interacts with permeant cations. The block is relieved at positive potentials, indicating that under these conditions Mg(2+) is able to permeate the selectivity filter of the channel. Although sizeable outward monovalent currents were recorded in the absence of intracellular Mg(2+), outward conductance is still approximately 10 times lower than inward conductance under symmetric, divalent-free ionic conditions. This Mg(2+)-independent rectification was preserved in inside-out patches and not altered by high intracellular concentrations of spermine, indicating that TRPV6 displays intrinsic rectification. Neutralization of a single aspartate residue within the putative pore loop abolished the Mg(2+) sensitivity of the channel, yielding voltage-independent, moderately inwardly rectifying monovalent currents in the presence of intracellular Mg(2+). The effects of intracellular Mg(2+) on TRPV6 are partially reminiscent of the gating mechanism of inwardly rectifying K(+) channels and may represent a novel regulatory mechanism for TRPV6 function in vivo.     

尼氟灭酸对肝癌细胞增殖的影响

为了观察氯通道阻断剂尼氟灭酸(NFA)对人肝癌细胞(kuman hepatoma cell,HHCC)增殖的影响,我们将NFA作用于HHCC,应用细胞计数法及噻唑兰(MTT)比色分析法观察细胞增殖情况;用流式细胞仪检测细胞周期时相;并用激光扫描共聚焦显微镜检测[Ca^2 ]i的变化。结果发现,NFA使HHCC细胞数及MTT光吸收值(OD)较对照组都显著降低,去除NFA后,OD值逐渐恢复。经100μmol/L NFA处理48h的HHCC细胞G1期细胞比例比对照组明显增高,S期及G2期细胞比例明显低于对照组。细胞外应用NFA(100μmol/L)使[Ca^2 ]i快速降低,去除NFA后,[Ca^2 ]i可恢复。这些结果表明,尼氟灭酸能抑制细胞增殖,其机制可能与细胞内信号转导Ca^2 /CaM途径被抑制有关。     

Flufenamic acid, mefenamic acid and niflumic acid inhibit single nonselective cation channels in the rat exocrine pancreas.

The non-steroidal anti-inflammatory drugs, flufenamic acid, mefenamic acid and niflumic acid, block Ca2(+)-activated non-selective cation channels in inside-out patches from the basolateral membrane of rat exocrine pancreatic cells. Half-maximal inhibition was about 10 microM for flufenamic acid and mefenamic acid, whereas niflumic acid was less potent (IC50 about 50 microM). Indomethacin, aspirin, diltiazem and ibuprofen (100 microM) had not effect. It is concluded that the inhibitory effect of flufenamate, mefenamate and niflumate is dependent on the specific structure, consisting of two phenyl rings linked by an amino bridge.     

Inhibition of thrombin,an unexplored function of retinoic acid

Retinoic acid, a derivative of vitamin A, is known to possess in vivo anti-inflammatory, anti-platelet and fibrinolytic activities. We have investigated the in vitro thrombin and platelet aggregation inhibitory activities of vitamin A (retinol) and its derivatives, retinoic acid and retinaldehyde. The thrombin enzymatic assay was performed fluorimetrically to assess the inhibition of thrombin (Sigma and plasma). Retinoic acid, retinaldehyde and retinol exhibited potent inhibition of thrombin, with IC₅₀ values of 67μg/ml, 74μg/ml and 152μg/ml, respectively for the inhibition of thrombin (Sigma); and 49μg/ml, 74μg/ml and 178μg/ml, respectively for the inhibition of thrombin (plasma). Amongst vitamin A and its derivatives, retinoic acid showed the highest inhibition of both the forms of thrombin. Vitamin A and its derivatives also displayed remarkable inhibition of platelet aggregation. This is the first report of vitamin A and its derivatives showing inhibition of thrombin and platelet aggregation in vitro.     

Molecular basis of ion selectivity, block, and rectification of the inward rectifier Kir3.1/Kir3.4 K(+) channel

The glycine-tyrosine-glycine (GYG) sequence in the p-loop of K+ channel subunits lines a narrow pore through which K+ ions pass in single file intercalated by water molecules. Mutation of the motif can give rise to non-selective channels, but it is clear that other structural features are also required for selectivity because, for instance, a recently identified class of cyclic nucleotide-gated pacemaker channels has the GYG motif but are poorly K+ selective. We show that mutation of charged glutamate and arginine residues behind the selectivity filter in the Kir3.1/Kir3.4 K+ channel reduces or abolishes K+ selectivity, comparable with previously reported effects in the Kir2.1 K+ channel. It has been suggested that a salt bridge exists between the glutamate-arginine residue pair. Molecular modeling indicates that the salt bridge does exist, and that it acts as a "bowstring" to maintain the rigid bow-like structure of the selectivity filter and restrict selectivity to K+. The modeling shows that relaxation of the bowstring by mutation of the residue pair leads to enhanced flexibility of the p-loop, allowing permeation of other cations, including polyamines. In experiments, mutation of the residue pair can also abolish polyamine-induced inward rectification. The latter effect occurs because polyamines now permeate rather than block the channel, to the remarkable extent that large polyamine currents can be measured.     

Inhibition of muscle glycogen phosphorylase b by 5-methyltetrahydrofolic acid, 3'-chloro- and 3',6'-dichloromethotrexates

Inhibition of rabbit skeletal muscle glycogen phosphorylase b by 5-methyl-5,6,7,8-tetrahydrofolic acid, 3'-chloro- and 3',5'-dichloromethotrexates has been studied. The inhibition is reversible and characterized by positive kinetic cooperativity (Hill coefficient exceeds 1). The values of pterin concentration causing two-fold diminishing of the enzymatic reaction rate increased in the order: 3',5'-dichloromethotrexate, 3'-chloromethotrexate, 5-methyl-5,6,7,8-tetrahydrofolic acid (0.24, 0.40 and 1.87 mM, respectively). Comparison of "half-saturation" concentrations for the above compounds and for methotrexate and folinic acid shows that pterin affinity to glycogen phosphorylase b is affected by substituents both in pteridine and in p-aminobenzoic moieties of the pterin molecule. The antagonism between 5-methyl-5,6,7,8-tetrahydrofolic acid, 3'-chloro- and 3',5'-dichloromethotrexates, on the one hand, and AMP and FMN, on the other, is revealed for combined action of modifiers on glycogen phosphorylase b.