The mechanism of inward rectification of potassium channels: "long-pore plugging" by cytoplasmic polyamines

The mechanism of inward rectification was examined in cell-attached and inside-out membrane patches from Xenopus oocytes expressing the cloned strong inward rectifier HRK1. Little or no outward current was measured in cell-attached patches. Inward currents reach their maximal value in two steps: an instantaneous phase followed by a time-dependent "activation" phase, requiring at least two exponentials to fit the time- dependent phase. After an activating pulse, the quasi-steady state current-voltage (I-V) relationship could be fit with a single Boltzmann equation (apparent gating charge, Z = 2.0 +/- 0.1, n = 3). Strong rectification and time-dependent activation were initially maintained after patch excision into high [K+] (K-INT) solution containing 1 mM EDTA, but disappeared gradually, until only a partial, slow inactivation of outward current remained. Biochemical characterization (Lopatin, A. N., E. N. Makhina, and C. G. Nichols, 1994. Nature. 372:366-396.) suggests that the active factors are naturally occurring polyamines (putrescine, spermidine, and spermine). Each polyamine causes reversible, steeply voltage-dependent rectification of HRK1 channels. Both the blocking affinity and the voltage sensitivity increased as the charge on the polyamine increased. The sum two Boltzmann functions is required to fit the spermine and spermidine steady state block. Putrescine unblock, like Mg2+ unblock, is almost instantaneous, whereas the spermine and spermidine unblocks are time dependent. Spermine and spermidine unblocks (current activation) can each be fit with single exponential functions. Time constants of unblock change e-fold every 15.0 +/- 0.7 mV (n = 3) and 33.3 +/- 6.4 mV (n = 5) for spermine and spermidine, respectively, matching the voltage sensitivity of the two time constants required to fit the activation phase in cell-attached patches. It is concluded that inward rectification in intact cells can be entirely accounted for by channel block. Putrescine and Mg2+ ions can account for instantaneous rectification; spermine and spermidine provide a slower rectification corresponding to so-called intrinsic gating of inward rectifier K channels. The structure of spermine and spermidine leads us to suggest a specific model in which the pore of the inward rectifier channel is plugged by polyamines that enter deeply into the pore and bind at sites within the membrane field. We propose a model that takes into account the linear structure of the natural polyamines and electrostatic repulsion between two molecules inside the pore. Experimentally observed instantaneous and steady state rectification of HRK1 channels as well as the time-dependent behavior of HRK1 currents are then well fit with the same set of parameters for all tested voltages and concentrations of spermine and spermidine.     

Intrinsic electrostatic potential in the BK channel pore: role in determining single channel conductance and block

The internal vestibule of large-conductance Ca(2+) voltage-activated K(+) (BK) channels contains a ring of eight negative charges not present in K(+) channels of lower conductance (Glu386 and Glu389 in hSlo) that modulates channel conductance through an electrostatic mechanism (Brelidze, T.I., X. Niu, and K.L. Magleby. 2003. Proc. Natl. Acad. Sci. USA. 100:9017-9022). In BK channels there are also two acidic amino acid residues in an extracellular loop (Asp326 and Glu329 in hSlo). To determine the electrostatic influence of these charges on channel conductance, we expressed wild-type BK channels and mutants E386N/E389N, D326N, E329Q, and D326N/E329Q channels on Xenopus laevis oocytes, and measured the expressed currents under patch clamp. Contribution of E329 to the conductance is negligible and single channel conductance of D326N/E329Q channels measured at 0 mV in symmetrical 110 mM K(+) was 18% lower than the control. Current-voltage curves displayed weak outward rectification for D326N and the double mutant. The conductance differences between the mutants and wild-type BK were caused by an electrostatic effect since they were enhanced at low K(+) (30 mM) and vanished at high K(+) (1 M K(+)). We determine the electrostatic potential change, Deltaphi, caused by the charge neutralization using TEA(+) block for the extracellular charges and Ba(2+) for intracellular charges. We measured 13 +/- 2 mV for Deltaphi at the TEA(+) site when turning off the extracellular charges, and 17 +/- 2 mV for the Deltaphi at the Ba(2+) site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined Deltaphi using a BK channel molecular model embedded in a lipid bilayer and solving the Poisson-Boltzmann equation. The model explains the experimental results adequately and, in particular, gives an economical explanation to the differential effect on the conductance of the neutralization of charges D326 and E329.     

Probing the geometry of the inner vestibule of BK channels with sugars

The geometry of the inner vestibule of BK channels was probed by examining the effects of different sugars in the intracellular solution on single-channel current amplitude (unitary current). Glycerol, glucose, and sucrose decreased unitary current through BK channels in a concentration- and size-dependent manner, in the order sucrose > glucose > glycerol, with outward currents being reduced more than inward currents. The fractional decrease of outward current was more directly related to the fractional hydrodynamic volume occupied by the sugars than to changes in osmolality. For concentrations of sugars < or =1 M, the i/V plots for outward currents in the presence and absence of sugar superimposed after scaling, and increasing K(+)(i) from 150 mM to 2 M increased the magnitudes of the i/V plots with little effect on the shape of the scaled curves. These observations suggest that sugars < or =1 M reduce outward currents mainly by entering the inner vestibule and reducing the movement of K(+) through the vestibule, rather than by limiting diffusion-controlled access of K(+) to the vestibule. With 2 M sucrose, the movement of K(+) into the inner vestibule became diffusion limited for 150 mM K(+)(i) and voltages > +100 mV. Increasing K(+)(i) then relieved the diffusion limitation. An estimate of the capture radius based on the 5 pA diffusion-limited current for channels without the ring of negative charge at the entrance to the inner vestibule was 2.2 A. Adding the radius of a hydrated K(+) (6-8 A) then gave an effective radius for the entrance to the inner vestibule of 8-10 A. Such a functionally wide entrance to the inner vestibule together with our observation that even small concentrations of sugar in the inner vestibule reduce unitary current suggest that a wide inner vestibule is required for the large conductance of BK channels.     

Quantitation of cation binding to wheat germ ribosomes: influences on subunit association equilibria and ribosome activity.

The binding of Mg2+, spermine, and spermidine to wheat germ ribosomes was quantitated following equilibrium dialysis. The Mg2+ binding data demonstrate that Mg2+ and K+ compete for binding to the ribosomes. Mg2+ binding saturates at approximately 0.56 positive charges per phosphate (+/P). The Mg2+, spermine and spermidine binding data indicate that either polyamine replaces Mg2+ upon binding to the ribosomes. Mg2+ and polyamine binding combined saturates at approximately 0.29 +/P under the conditions reported. When a critical number of Mg2+ ions are replaced by either polyamine, the activity of the ribosomes falls dramatically. Ribosomal subunit association increases with the degree of phosphate charge neutralization due to the binding of Mg2+. Total charge neutralization during subunit association by Mg2+ and polyamine binding combined, is much less than that achieved by Mg2+ alone.     

Rectification of rabbit cardiac ryanodine receptor current by endogenous polyamines.

The actions of three endogenous polyamines (spermine, spermidine, and putrescine) were defined on Ca2+ release channels (ryanodine receptors, RyRs) isolated from rabbit cardiac sarcoplasmic reticulum. The current-voltage relationship of the RyR channel was N-shaped in the presence of polyamine (1-5 mM). Polyamine blocked conduction near 0 mV, but the blockade was relieved at large potentials. Polyamines acted (blocked) from both sides of the channel. Polyamine efficacy was dependent on current direction and was inversely related to the ion selectivity of the RyR pore. This suggests that polyamine interacts with current-carrying ions in the permeation pathway. The apparent half-block concentration of spermine at 0 mV was < 0.1 mM. The features of polyamine blockade suggest that the polyamines are permeable cationic blockers of the RyR channel. Further, the levels of polyamines found in muscle cells are sufficient to block single RyR channels and thus may alter the sarcoplasmic reticulum Ca2+ release process in situ.     

Effect of polyamines on Mg(2+)-dependent ATP-hydrolase activity in plasmatic membrane of myometrium cells

Influence of aliphatic polyamines of spermine and spermidine on the enzymatic activity of the ouabain-sensitive Na+,K(+)-ATPase and the ouabain-resistant basal Mg(2+)-ATPase (specific activity--10.6 +/- 0.9 and 18.1 +/- 1.2 microM P(i)/hour on 1 mg of protein accordingly, n = 7) has been studied in the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution. It was found, that the polyamine spermine in concentration of 1 and 10 mM activated the Na+,K(+)-ATPase by 54 and 64% on the average relative to control value. Spermidine also stimulated the Na+,K(+)-ATPase activity, however it did it less efficiently than spermine: by 8 and 20% on the average at concentration of 1 and 10 mM, accordingly. Similarly, polyamines had affect on the basal Mg(2+)-ATPase: spermine in concentration of 1 and 10 mM activated it by 26 and 39% relative to control value; spermidine in concentration of 1 and 10 mM activated it by 10 and 32% relative to control. The magnitudes of the apparent activation constant K(a) of spermine were 0.35 +/- 0.07 and 0.10 +/- 0.02 mM for Na+,K(+)-ATPase and basal Mg(2+)-ATPase, accordingly (M +/- m, n = 5). It is supposed, that the obtained experimental data can be useful in the further research of the membrane mechanisms underlying of the cationic exchange in the smooth muscles, in particular, when investigating the role of the plasmatic membrane in providing electromechanical coupling in them, and also in regulation of ionic homeostasis in the smooth muscle cells.     

Novel gating mechanism of polyamine block in the strong inward rectifier K channel Kir2.1

Inward rectifying K channels are essential for maintaining resting membrane potential and regulating excitability in many cell types. Previous studies have attributed the rectification properties of strong inward rectifiers such as Kir2.1 to voltage-dependent binding of intracellular polyamines or Mg to the pore (direct open channel block), thereby preventing outward passage of K ions. We have studied interactions between polyamines and the polyamine toxins philanthotoxin and argiotoxin on inward rectification in Kir2.1. We present evidence that high affinity polyamine block is not consistent with direct open channel block, but instead involves polyamines binding to another region of the channel (intrinsic gate) to form a blocking complex that occludes the pore. This interaction defines a novel mechanism of ion channel closure.     

Spermine block of the strong inward rectifier potassium channel Kir2.1: dual roles of surface charge screening and pore block

Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg(2+). Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers.     

Low resistance, large dimension entrance to the inner cavity of BK channels determined by changing side-chain volume

Large-conductance Ca²⁺- and voltage-activated K⁺ (BK) channels have the largest conductance (250–300 pS) of all K⁺-selective channels. Yet, the contributions of the various parts of the ion conduction pathway to the conductance are not known. Here, we examine the contribution of the entrance to the inner cavity to the large conductance. Residues at E321/E324 on each of the four α subunits encircle the entrance to the inner cavity. To determine if 321/324 is accessible from the inner conduction pathway, we measured single-channel current amplitudes before and after exposure and wash of thiol reagents to the intracellular side of E321C and E324C channels. MPA⁻ increased currents and MTSET⁺ decreased currents, with no difference between positions 321 and 324, indicating that side chains at 321/324 are accessible from the inner conduction pathway and have equivalent effects on conductance. For neutral amino acids, decreasing the size of the entrance to the inner cavity by substituting large side-chain amino acids at 321/324 decreased outward single-channel conductance, whereas increasing the size of the entrance with smaller side-chain substitutions had little effect. Reductions in outward conductance were negated by high [K⁺]_i. Substitutions had little effect on inward conductance. Fitting plots of conductance versus side-chain volume with a model consisting of one variable and one fixed resistor in series indicated an effective diameter and length of the entrance to the inner cavity for wild-type channels of 17.7 and 5.6 Å, respectively, with the resistance of the entrance ∼7% of the total resistance of the conduction pathway. The estimated dimensions are consistent with the structure of MthK, an archaeal homologue to BK channels. Our observations suggest that BK channels have a low resistance, large entrance to the inner cavity, with the entrance being as large as necessary to not limit current, but not much larger.     

Estimation of polyamine binding to macromolecules and ATP in bovine lymphocytes and rat liver.

To estimate the polyamine distribution in bovine lymphocytes and rat liver, the binding constants (K) for DNA, RNA, phospholipid, and ATP were determined under the conditions of 10 mM Tris-HCl, pH 7.5, 2 mM Mg2+, and 150 mM K+. The binding constants of spermine for calf thymus DNA, Escherichia coli 16 S rRNA, phospholipid in rat liver microsomes and ATP were 1.15 x 10(2), 6.69 x 10(2), 2.22 x 10(2), and 5.95 x 10(2) M-1, respectively. From these binding constants and experimentally determined cellular concentrations of macromolecules, ATP, and polyamines, spermine distribution in the cells was estimated. In bovine lymphocytes, the mols of spermine bound to DNA, RNA, phospholipid, and ATP were 0.79, 3.7, 0.23, and 4.3 per 100 mol of phosphate of macromolecules or ATP, respectively. In rat liver, they were 0.19, 1.0, 0.05, and 0.97/100 mol of phosphate of macromolecules or ATP, respectively. The binding constants of spermidine for macromolecules and ATP were smaller than those of spermine, but a similar tendency was observed with spermidine distribution among macromolecules and ATP in the above two cells. The amount of polyamine bound to DNA and phospholipid was significantly lower than that to RNA. When either the Mg2+ or K+ concentration increased, the amount of free spermine and that bound to RNA and ATP increased, but the amount of spermine bound to DNA and phospholipid decreased. The results indicate that most polyamines exist as a polyamine-RNA complex in cells. Under the conditions that globin synthesis is stimulated by spermine in a rabbit reticulocyte cell-free system, the amount of spermine bound to RNA was very close to the value estimated in the cells.     

Modulation of neuronal voltage-activated calcium and sodium channels by polyamines and pH

The endogenous polyamines spermine, spermidine and putrescine are present at high concentrations inside neurons and can be released into the extracellular space where they have been shown to modulate ion channels. Here, we have examined polyamine modulation of voltage-activated Ca(2+) channels (VACCs) and voltage-activated Na(+) channels (VANCs) in rat superior cervical ganglion neurons using whole-cell voltage-clamp at physiological divalent concentrations. Polyamines inhibited VACCs in a concentration-dependent manner with IC(50)s for spermine, spermidine, and putrescine of 4.7 +/- 0.7, 11.2 +/- 1.4 and 90 +/- 36 mM, respectively. Polyamines caused inhibition by shifting the VACC half-activation voltage (V(0.5)) to depolarized potentials and by reducing total VACC permeability. The shift was described by Gouy-Chapman-Stern theory with a surface charge density of 0.120 +/- 0.005 e(-) nm(-2) and a surface potential of -19 mV. Attenuation of spermidine and spermine inhibition of VACC at decreased pH was explained by H(+) titration of surface charge. Polyamine-mediated effects also decreased at elevated pH due to the inhibitors having lower valence and being less effective at screening surface charge. Polyamines affected VANC currents indirectly by reducing TTX inhibition of VANCs at high pH. This may reflect surface charge induced decreases in the local TTX concentration or polyamine-TTX interactions. In conclusion, polyamines inhibit neuronal VACCs via complex interactions with extracellular H(+) and Ca. Many of the observed effects can be explained by a model incorporating polyamine binding, H(+) binding and surface charge screening.     

C-terminus determinants for Mg2+ and polyamine block of the inward rectifier K+ channel IRK1.

Critical loci for ion conduction in inward rectifier K+ channels are only now being discovered. The C-terminal region of IRK1 plays a crucial role in Mg2+i blockade and single-channel K+ conductance. A negatively charged aspartate in the putative second transmembrane domain (position 172) is essential for time-dependent block by the cytoplasmic polyamines spermine and spermidine. We have now localized the C-terminus effect in IRK1 to a single, negatively charged residue (E224). Mutation of E224 to G, Q and S drastically reduced rectification. Furthermore, the IRK1 E224G mutation decreased block by Mg2+i and spermidine and, like the E224Q mutation, caused a dramatic reduction in the apparent single-channel K+ conductance. The double mutation IRK1 D172N+ E224G was markedly insensitive to spermidine block, displaying an affinity similar to ROMK1. The results are compatible with a model in which the negatively charged residue at position 224, E224, is a major determinant of pore properties in IRK1. By means of a specific interaction with the negatively charged residue at position 172, D172, E224 contributes to the formation of the binding pocket for Mg2+ and polyamines, a characteristic of strong inward rectifiers.     

Effects of Polyamines on Proline Endopeptidase Activity in Rat Brain

The in vitro effects of polyamines on the activity of proline endopeptidase (PEPase) in rat brain cytosol, which contains an endogenous PEPase inhibitor, have been studied. Of the three amines tested (spermine, spermidine, and putrescine), spermine and spermidine markedly enhanced the enzyme activity in brain cytosol. At 6.25 mM spermine or 25 mM spermidine, a 13- or 14-fold enhancement of the enzyme activity was observed. When Mg2+ was used, an approximately fourfold enhancement of the enzyme activity was observed at 50 mM. The enhancement produced by spermine or spermidine was unaffected by Mg2+ up to 50 mM. The activity of purified PEPase was only slightly affected by each polyamine, but it was inhibited 50% by 50 mM Mg2+. On the other hand, 50% inhibition of the enzyme produced by the purified PEPase inhibitor (Mr 7,000: Ki 0.67 mM) was completely restored by addition of 0.7 mM spermine, 3.5 mM spermidine, or 28 mM putrescine. This restoration of inhibition by polyamines was reversed by increasing the inhibitor concentration. These data suggest that polyamines effectively reverse the inhibition of PEPase by its endogenous inhibitor by the reversible formation of a kinetically significant complex. The possible functions of polyamines in the regulation of PEPase in vivo are discussed.     

Polyamines secreted by cancer cells possibly account for the impairment of the human erythrocyte sodium pump activity.

Using an original microcalorimetric method, we previously showed that in erythrocytes from cancer patients, the sodium pump activity was decreased and returned to normal in patient in remission. In addition we suggested that a plasma-borne factor probably secreted by cancer cells accounted for this impairment of the sodium transporter. In the present study we sought to identify this factor as well as its mechanism of action. First we determined the effect of culture media from undifferentiated and differentiated colon cancer cell lines (Caco-2 and HT29-D4) on the sodium pump activity of normal human erythrocytes. The inhibitory powers of culture media from undifferentiated cells were higher than those of differentiated cells (38.6 +/- 3.5% vs 6.9 +/- 4.6%, p<0.05 for Caco-2 and 45.8 +/- 6.2% vs 9.0 +/- 5.0%, <0.05 for HT29-D4). The use of alpha difluoro-methylomithine (2 mM) to inhibit ornithine decarboxylase, the rate-limiting enzyme for polyamine biosynthesis, dramatically reduced the sodium pump inhibition induced by the two undifferentiated cell lines (75% for Caco-2 and 89% for HT29-D4). Polyamines secreted by undifferentiated cells and then taken up by human erythrocytes thus appeared as inhibitors of sodium pump of these red blood cells. Putrescine, spermidine, and spermine (the main polyamines) exerted a similar inhibitory effect (33 +/- 2%). Tested in vitro on Na,KATPase, these polyamines (3 mM) were inhibitors (putrescine = 23 +/- 2%; spermidine= 48 +/- 3%; spermine= 55 +/- 2%) when assay condition for the ATPase reaction was suboptimal (Na+ = 10 mM; K+ = 1 mM). The inhibitory effect appeared to be related to their charge and their aliphatic chain length. The effect of spermidine and spermine on the ionic substrates and ATP-Mg showed that molecules decreased the affinity (Km) of the Na,K-ATPase for Na+ (11.24 +/- 0.49 mM for control vs 23.51 +/- 1.53 mM for spermine and 18.86 +/- 0.98 mM for spermidine), indicating that polyamines exerted their inhibitory effect in a competitive manner.     

Modulation of Neuronal Voltage-Activated Calcium and Sodium Channels by Polyamines and pH

The endogenous polyamines spermine, spermidine and putrescine are present at high concentrations inside neurons and can be released into the extracellular space where they have been shown to modulate ion channels. Here, we have examined polyamine modulation of voltage-activated Ca2+ channels (VACCs) and voltage-activated Na+ channels (VANCs) in rat superior cervical ganglion neurons using whole-cell voltage-clamp at physiological divalent concentrations. Polyamines inhibited VACCs in a concentration-dependent manner with IC50s for spermine, spermidine, and putrescine of 4.7 ± 0.7, 11.2 ± 1.4, and 90 ± 36 mM, respectively. Polyamines caused inhibition by shifting the VACC half-activation voltage (V0.5) to depolarized potentials and by reducing total VACC permeability. The shift was described by Gouy-Chapman-Stern theory with a surface charge density of 0.120 ± 0.005 e- nm-2 and a surface potential of -19 mV. Attenuation of spermidine and spermine inhibition of VACC at decreased pH was explained by H+ titration of surface charge. Polyamine-mediated effects also decreased at elevated pH due to the inhibitors having lower valence and being less effective at screening surface charge. Polyamines affected VANC currents indirectly by reducing TTX inhibition of VANCs at high pH. This may reflect surface charge induced decreases in the local TTX concentration or polyamine-TTX interactions. In conclusion, polyamines inhibit neuronal VACCs via complex interactions with extracellular H+ and Ca. Many of the observed effects can be explained by a model incorporating polyamine binding, H+ binding and surface charge screening.     

Effect of spermine on activity of purified Ca2+, Mg2+-ATPase from plasma membranes of myometrium cells

Effect of endogenous polyamine spermine, a relaxant of smooth muscle, on the activity of myometrium cell plasma membrane Ca2+, Mg(2+)-ATPase was studied. It was observed a tendency to activation of enzyme at the spermine concentrations 0.1-0.5 mM, the increase of the polyamine concentrations up to 10 mM inhibited. ATPase by 80% (I50 = 5.5 +/- 0.3 mM). Spermine inhibited enzyme decreasing its turnover rate and affinity for Ca2+. The ATPase affinity for Mg2+ increased in the presence of spermine. It was revealed, that the inhibitory effect of spermine is changed by the stimulatory effect under the increase of Ca2+ concentration (up to 2.6 microM), that correlates with the relaxing effect of this polyamine on the smooth muscle.     

Cytoplasmic polyamines block the fast-activating vacuolar cation channel

The fast-activating vacuolar (FV) channel dominates the electrical characteristics of the tonoplast at physiological free Ca²⁺ concentrations. Since polyamines are known to increase in plant cells in response to stress, the regulation of FV channels by polyamines was investigated. Patch-clamp measurements were performed on whole barley ( Hordeum vulgare ) mesophyll vacuoles and on excised tonoplast patches. The trivalent polyamine spermidine and the tetravalent polyamine spermine blocked FV channels with K_d≈ 100 μM and K_d≈ 5 μM, respectively. Increasing cytosolic and vacuolar Ca²⁺ had no effect on putrescine and spermidine binding to FV channels but slightly decreased the affinity for spermine. The inhibition of FV channels by all three polyamines was not voltage-dependent. This points to a different mode of binding compared to inward rectifier K⁺ channels and Ca²⁺-permeable glutamate receptor channels from animal cells, which show rectification due to a voltage-dependent block by polyamines. In plant cells, the common polyamines (putrescine, spermidine and spermine) are likely to mediate a salt stress-induced decrease of ion flux across the vacuolar membrane by blocking FV channels.     

The binding of polyamines and magnesium to DNA.

1. The binding of spermine and Mg2+ to DNA has been investigated using the dye arsenazo III to measure unbound cations. 2. The apparent dissociation constant, Kd, of DNA for spermine has been found to be 7.4 +/- 3.9 x 10(-8) M and that for Mg2+, 6.5 +/- 3.3 x 10(-7) M. 3. Binding of spermine in the presence of 1 mM Mg2+ has been shown to have a Kd of about 4 x 10(-6) M. 4. Magnesium ion (2 mM) halves the concentration of spermine needed to cause DNA aggregation. 5. Spermidine binds to DNA with a similar affinity to spermine but 3,3'-iminobispropylamine and 1,5,9,13-tetra-azatridecane bind with a lower affinity. The naturally occurring polyamines thus have a higher affinity for DNA than the related polyamines which do not occur naturally. 6. Binding of spermine or spermidine to DNA alters the spectrophotometric absorbance of DNA at 260 nm.     

Regulation of the F-ATPase from mitochondria of Vigna sinensis (L.) Savi cv. Pitiuba by spermine, spermidine, putrescine, Mg2+, Na+, and K+

Mitochondria from Vigna sinensis (L.) Savi cv. Pitiuba contain the polyamines spermine, spermidine, and putrescine. The membrane-bound F1-ATPase from mitochondria of Vigna sinensis is activated by these polyamines at physiological concentrations. The effect of polyamines on the membrane-bound of F1-ATPase is dependent on the concentrations of Na+, K+, MgATP, and Mg2+. Excess Na+ or K+ prevents the activation of the membrane-bound F1-ATPase by spermine and spermidine, but not by putrescine. The most pronounced effects were observed at low MgATP concentrations in the absence of Na+ and K+. At [MgATP] = 0.08 mM, spermine activation of the membrane-bound F1-ATPase was 130%. The membrane-bound F1-ATPase is slightly activated by Mg2+ at lower concentrations and strongly inhibited by Mg2+ at higher concentrations. Activation as well as inhibition is dependent on the substrate MgATP concentration. Although there is competition between Mg2+ and MgATP, the binding sites for these two ligands are different (pseudocompetitive inhibition). The inhibition of the membrane-bound F1-ATPase can be reversed by polyamines. There is evidence that the binding sites for Mg2+ and polyamines are identical. The F1-ATPase detached from the membrane is neither activated by polyamines nor inhibited by Mg2+. Therefore, the binding sites for Mg2+ and polyamines seem to be localized on the membrane.     

Protons block BK channels by competitive inhibition with K+ and contribute to the limits of unitary currents at high voltages

Proton block of unitary currents through BK channels was investigated with single-channel recording. Increasing intracellular proton concentration decreased unitary current amplitudes with an apparent pKa of 5.1 without discrete blocking events, indicating fast proton block. Unitary currents recorded at pH(i) 8.0 and 9.0 had the same amplitudes, indicating that 10(-8) M H(+) had little blocking effect. Increasing H(+) by recording at pH(i) 7.0, 6.0, and 5.0 then reduced the unitary currents by 13%, 25%, and 53%, respectively, at +200 mV. Increasing K(+)(i) relieved the proton block in a manner consistent with competitive inhibition of K(+)(i) action by H(+)(i). Proton block was voltage dependent, increasing with depolarization, indicating that block was coupled to the electric field of the membrane. Proton block was not described by the Woodhull equation for noncompetitive voltage-dependent block, but was described by an equation for cooperative competitive inhibition that included voltage-dependent block from the Woodhull equation. Proton block was still present after replacing the eight negative charges in the ring of charge at the entrance to the intracellular vestibule by uncharged amino acids. Thus, the ring of charge is not the site of proton block or of competitive inhibition of K(+)(i) action by H(+)(i). With 150 mM symmetrical KCl, unitary current amplitudes increased with depolarization, reaching 66 pA at +350 mV (pH(i) 7.0). The increase in amplitude with voltage became sublinear for voltages >100 mV. The sublinearity was unaffected by removing from the intracellular solutions Ca(2+) and Ba(2+) ions, the Ca(2+) buffers EGTA and HEDTA, the pH buffer TES, or by replacing Cl(-) with MeSO(3)(-). Proton block accounted for approximately 40% of the sublinearity at +200 mV and pH 7.0, indicating that factors in addition to proton block contribute to the sublinearity of the unitary currents through BK channels.