Binding of alkaline cations to the double-helical form of gramicidin.

Gramicidin is a polypeptide antibiotic that forms monovalent cation-specific channels in membrane environments. In organic solvents and in lipids containing unsaturated fatty acid chains, it forms a double-helical "pore" structure, in which two monomers are intertwined. This form of gramicidin can bind two cations inside its lumen, and the crystal structures of both an ion complex and an ion-free form have been determined. In this study, we have used circular dichroism (CD) spectroscopy to examine the binding mechanism and the binding constants (K1 and K2) of cations to gramicidin in the double helical form in methanol solution. The dramatic change in optical rotation in the far-ultraviolet CD spectrum of gramicidin provides a useful tool for monitoring the binding. The binding mechanism appears to involve a large conformation change associated with the binding of ions to the first of the two sites. The calculated values for the K1 binding constants for alkaline cations are considerably smaller than the K2 binding constants. The order of binding affinity for alkaline cations is similar to that for the helical dimer "channel" form of gramicidin, i.e., Cs+ approximately Rb+ > > K+ > Li+, but in comparison to the helical dimer form, the binding to double-helical dimers is dominated by a cation size-dependent conformational change in the gramicidin structure.     

Thermodynamic parameters for the binding of divalent cations to gramicidin A incorporated into a lipid environment by Tl-205 nuclear magnetic resonance.

Thermodynamic parameters, enthalpy and entropy, for the binding of the divalent cations, Mg+2, Ca+2, Sr+2, Ba+2, and Cd+2, to gramicidin A, incorporated into lysophosphatidylcholine, have been determined using a combination of Tl-205 nuclear magnetic resonance spectroscopy and competition binding. The binding process is thermodynamically driven by the enthalpy and not the entropy. The enthalpy values are related to the process involving the transfer of cations from an aqueous environment to an amide environment. A comparison is made between the thermodynamic parameters for the binding of monovalent and divalent cations to gramicidin A to illustrate the channel blocking ability of the divalent cations with respect to monovalent cation transport.     

Heavy metal cation-induced increase in the antimicrobial activity of gramicidin S. Increased sensitivity of metal-resistant mutants of Escherichia coli B to the antibiotic]

Gramicidin S response of metal resistant mutants of E. coli B and the effect of concentrations of Cu2+, Ag+, Co2+ and Cd2+ on the growth and sensitivity of E. coli B to cationic antibiotics, i.e. gramicidin S2+ and streptomycin2+, were studied. It was shown that the metal-cumulating mutants of E. coli B with two different mechanisms of cross resistance to Cu2+, Cd2+ and Ag+ had higher sensitivity to gramicidin S than the initial wild type strain of E. coli B. It was found that in the threshold or higher doses the salts of Cu, Ag, Co and Cd increased the gramicidin S antimicrobial action on actively metabolizing cells of E. coli B. Analysis of the experimental data as well as the literature ones suggested that the synergic action of gramicidin S and the heavy metals stemmed from an increase in the cationic conductivity of the cytoplasma membrane modified by the metals in the threshold doses which induced an increase in the transport and accumulation of the cations in the bacterial cells by the electric field gradient (with the negative sign inside). Withdrawal of Ca2+ and Mg2+ from the E. coli outer structures into the cytoplasm impaired the barrier properties of the outer membrane and promoted binding of the gramicidin S cations to the liberated anionic groups of the E. coli outer structures and potentiation of the gramicidin S antimicrobial activity as was shown in our experiments.     

Binding constants of Li+, K+, and Tl+ in the gramicidin channel determined from water permeability measurements.

In an open circuit there can be no net cation flux through membranes containing only cation-selective channels, because electroneutrality must be maintained. If the channels are so narrow that water and cations cannot pass by each other, then the net water flux through those "single-file" channels that contain a cation is zero. It is therefore possible to determine the cation binding constants from the decrease in the average water permeability per channel as the cation concentration in the solution is increased. Three different methods were used to determine the osmotic water permeability of gramicidin channels in lipid bilayer membranes. The osmotic water permeability coefficient per gramicidin channel in the absence of cations was found to be 6 x 10(-14) cm3/s. As the cation concentration was raised, the water permeability decreased and a binding constant was determined from a quantitative fit to the data. When the data were fitted assuming a maximum of one ion per channel, the dissociation constant was 115 mM for Li+, 69 mM for K+, and 2 mM for Tl+.     

Ion-specific diffusion rates through transmembrane protein channels. A molecular dynamics study

The migration of different alkali metal cations through a transmembrane model channel is simulated by means of the molecular dynamics technique. The parameters of the model are chosen in close relation to the gramicidin A channel. Coulomb- and van der Waals-type potentials between the ions and flexible carbonyl groups of the pore-forming molecule are used to describe the ion channel interaction. The diffusion properties of the ions are obtained from three-dimensional trajectory calculations. The diffusion rates for the different ions Li+, Na+, K+ and Rb+ are affected not only by the mass of the particles but also very strongly by their size. The latter effect is more pronounced for rigid channels, i.e., for binding vibrational frequencies of the CO groups with v greater than 400 cm-1. In this range the selectivity sequence for the diffusion rates is the inverse of that expected from normal rate theory but agrees with that found in experiments for gramicidin A.     

Location of ion-binding sites in the gramicidin channel by X-ray diffraction.

We report the first X-ray diffraction on gramicidin in its membrane-active form by using uniformly aligned multilayer samples of membranes containing gramicidin and ions (T1+, K+, Ba2+, Mg2+ or without ions). From the difference electron density profiles, we found a pair of symmetrically located ion-binding sites for T1- at 9.6 (+/- 0.3) A and for Ba2+ at 13.0 (+/- 0.2) A from the midpoint of the gramicidin channel. The location of Ba(2+)-binding sites is near the ends of the channel, consistent with the experimental observation that divalent cations do not permeate but block the channel. The location of T1(+)-binding sites is somewhat of a surprise. It was generally thought that monovalent cations bind to the first turn of the helix from the mouth of the channel. (It is now generally accepted that the gramicidin channel is a cylindrical pore formed by two monomers, each a single-stranded beta 6.3 helix and hydrogen-bonded head-to-head at their N termini.) But our experiment shows that the T1(+)-binding site is either near the bottom of or below the first helix turn.     

Ion transport in the gramicidin channel: molecular dynamics study of single and double occupancy.

The structural and thermodynamic factors responsible for the singly and doubly occupied saturation states of the gramicidin channel are investigated with molecular dynamics simulations and free energy perturbation methods. The relative free energy of binding of all of the five common cations Li+, Na+, K+, Rb+, and Cs+ is calculated in the singly and doubly occupied channel and in bulk water. The atomic system, which includes the gramicidin channel, a model membrane made of neutral Lennard-Jones particles and 190 explicit water molecules to form the bulk region, is similar to the one used in previous work to calculate the free energy profile of a Na+ ion along the axis of the channel. In all of the calculations, the ions are positioned in the main binding sites located near the entrances of the channel. The calculations reveal that the doubly occupied state is relatively more favorable for the larger ions. Thermodynamic decomposition is used to show that the origin of the trend observed in the calculations is due to the loss of favorable interactions between the ion and the single file water molecules inside the channel. Small ions are better solvated by the internal water molecules in the singly occupied state than in the doubly occupied state; bigger ions are solvated almost as well in both occupation states. Water-channel interactions play a role in the channel response. The observed trends are related to general thermodynamical properties of electrolyte solutions.     

Use of weak acids to determine the bulk diffusion limitation of H+ ion conductance through the gramicidin channel.

The addition of 2 M formic acid at pH 3.75 increased the single channel H+ ion conductance of gramicidin channels 12-fold at 200 mV. Other weak acids (acetic, lactic, oxalic) produce a similar, but smaller increase. Formic acid (and other weak acids) also blocks the K+ conductance at pH 3.75, but not at pH 6.0 when the anion form predominates. This increased H+ conductance and K+ block can be explained by formic acid (HF) binding to the mouth of the gramicidin channel (Km = 1 M) and providing a source of H+ ions. A kinetic model is derived, based on the equilibrium binding of formic acid to the channel mouth, that quantitatively predicts the conductance for different mixtures of H+, K+, and formic acid. The binding of the neutral formic acid to the mouth of the gramicidin channel is directly supported by the observation that a neutral molecule with a similar structure, formamide (and malonamide and acrylamide), blocks the K+ conductance at pH 6.0. The H+ conductance in the presence of formic acid provides a lower bound for the intrinsic conductance of the gramicidin channel when there is no diffusion limitation at the channel mouth. The 12-fold increase in conductance produced by formic acid suggests that greater than 90% of the total resistance to H+ results from diffusion limitation in the bulk solution.     

The effect of di- and trivalent metal ions on the binding of [3H-Tyr1, D-Ala2, D-Leu5] enkephalin to opiate receptors from the rat brain

The influence of Ca2+, Mg2+, Mn2+, Sr2+, La3+, Nd3+, Sm3+, Eu3+, and Gd3+ ions on the binding of labeled, stable enkephalin analogue, [3H-Tyr1, D-Ala2, D-Leu5]enkephalin, to opiate receptors of the rat brain membrane preparations has been investigated. The formation of the complex can be described by a scheme involving at least two independent binding sites. The high affinity site does not discriminate the divalent and trivalent metal ions: all examined cations enhanced the enkephalin affinity for this site. The ligand binding to the low affinity site is potentiated only by Mn2+, Mg2+, and lathanoides. The maximal concentration of the binding sites of the above two types is not affected by the cations. The increase in the ionic strength of the solution entails a decrease in the affinity of the ligand for the high affinity binding site. It is shown that the effect of both di- and trivalent metal cations on the [3H-Tyr1, D-Ala2, D-Leu3] enkephalin binding is mediated through one cation attachment site on the respective enkephalin receptor.     

Equilibrium binding constants for Tl+ with gramicidins A, B and C in a lysophosphatidylcholine environment determined by 205Tl nuclear magnetic resonance spectroscopy.

Nuclear Magnetic Resonance (NMR) 205Tl spectroscopy has been used to monitor the binding of Tl+ to gramicidins A, B, and C packaged in aqueous dispersions of lysophosphatidylcholine. For 5 mM gramicidin dimer in the presence of 100 mM lysophosphatidylcholine, only approximately 50% or less of the gramicidin appears to be accessible to Tl+. Analysis of the 205Tl chemical shift as a function of Tl+ concentration over the 0.65-50 mM range indicates that only one Tl+ ion can be bound by gramicidin A, B, or C under these experimental conditions. In this system, the Tl+ equilibrium binding constant is 582 +/- 20 M-1 for gramicidin 1949 +/- 100 M-1 for gramicidin B, and 390 +/- 20 M-1 for gramicidin C. Gramicidin B not only binds Tl+ more strongly but it is also in a different conformational state than that of A and C, as shown by Circular Dichroism spectroscopy. The 205Tl NMR technique can now be extended to determinations of binding constants of other cations to gramicidin by competition studies using a 205Tl probe.     

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.     

The divalent cation-binding sites of gramicidin A transmembrane ion-channel.

The conductance of the gramicidin A single channels in glycerolmonooleate membranes is strongly reduced in the presence of Mn2+ cations. The nmr experiments were performed for N-terminal to N-terminal gramicidin A dimer formed by two right-handed single-stranded helixes incorporated into the sodium dodecyl sulfate micelles in the presence of Mn2+ ions. Dependence of the nonselective spin-lattice relaxation rates of the gramicidin A protons on Mn2+ concentration was analyzed to determine coordinates of the divalent cation binding sites. It is inferred that Mn2+ ions are bound at the channel mouths at distances of 6.4, 8.6, and 8.8 A (+/- 2 A) from the oxygen atoms of exposed carbonyl groups of D-Leu 12, 14, and 10, respectively. The bounded Mn2+ retains its hydrate shell, the size of which (approximately 6 A) exceeds the inner pore diameter (approximately 4 A). That makes the gramicidin A channel impermeable for divalent cations.     

Cation-dependent reassembly of F0F1-ATPase in submitochondrial particles: evidence for a binding site for F1 on F0 in the absence of F6 and oligomycin sensitivity-conferring protein

Bovine heart submitochondrial particles depleted of F1, OSCP (oligomycin sensitivity-conferring protein), and F6 require the presence of cations to rebind F1. Among the cations tested, NH4+, Cs+, and Rb+ were most efficient, followed by K+, Na+, Li+, Ca2+, and Mg2+. The extent of F1 binding approached that occurring upon supplementation with F6 and/or OSCP, and was similar to the F1 content of particles prior to depletion. In the absence of cations, F6 and/or OSCP were ineffective in promoting the binding of F1 to the depleted particles. The F1 bound to the particles in the presence of cations alone was completely insensitive to oligomycin. It remained bound to the particles after removal of the cation, and could be rendered partially (approximately 50%) or maximally (less than 80%) oligomycin-sensitive upon the subsequent addition of OSCP or of F6 and OSCP, respectively. The surface potential of the particles, as determined by microelectrophoresis, was screened by all cations tested, regardless of their ability to promote the binding of F1; this was in contrast to earlier findings with particles depleted of F1 only, where the ability of cations to promote the rebinding of F1 paralleled their efficiency to neutralize the surface charge of the particle membrane. It is concluded that the effect of cations on the binding of F1 to F1-, F6-, and OSCP-depleted particles is due to a specific interaction of the cations with certain segments or components of the membrane. The results suggest the existence of a binding site for F1 on F0 in addition to the binding site(s) provided by F6 and OSCP.     

Equilibrium binding constants for the group I metal cations with gramicidin-A determined by competition studies and T1+-205 nuclear magnetic resonance spectroscopy.

The equilibrium binding constants of the Group I metal cations with gramicidin A in aqueous dispersions of lyso-PC have been determined using a combination of competitive binding with the T1+ ion and T1-205 NMR spectroscopy. The values of the binding constants at 34 degrees C are Li (32.2 M-1), Na (36.9 M-1), K (52.6 M-1), Rb (55.9 M-1), and Cs (54.0 M-1). The equilibrium binding constant for the T1+ ion at this temperature is 582 M-1. The relationships between the binding constants, the free energy of the binding process, and the cation selectivity of the gramicidin A channel are discussed.     

Quantitative analysis of monovalent counterion binding to random-sequence, double-stranded DNA using the replacement ion method

A variation of affinity capillary electrophoresis, called the replacement ion (RI) method, has been developed to measure the binding of monovalent cations to random sequence, double-stranded (ds) DNA. In this method, the ionic strength is kept constant by gradually replacing a non-binding ion in the solution with a binding ion and measuring the mobility of binding and non-binding analytes as a function of binding ion concentration. The method was validated by measuring the binding of Li+ ions to adenosine nucleotides; the apparent dissociation constants obtained by the RI method are comparable to literature values obtained by other methods. The binding of Tris+, NH4+, Li+, Na+, and K+ to dsDNA was then investigated. The apparent dissociation constants observed for counterion binding to a random-sequence 26-base pair (bp) oligomer ranged from 71 mM for Tris+ to 173 mM for Na+ and K+. Hence, positively charged Tris buffer ions will compete with other monovalent cations in Tris-buffered solutions. The bound cations identified in this study may correspond to the strongly correlated, tightly bound ions recently postulated to exist as a class of ions near the surface of dsDNA (Tan, Z.-J., and Chen, S.-J. (2006) Biophys. J. 91, 518-536). Monovalent cation binding to random-sequence dsDNA would be expected to occur in addition to any site-specific binding of cations to A-tracts or other DNA sequence motifs. Single-stranded DNA oligomers do not bind the five tested cations under the conditions investigated here.     

The stimulation by monovalent cations of the amidase activity of bovine des-1-41 light chain activated protein C

The kinetic properties of the activation by monovalent cations of the amidolytic activity of bovine des-1-41 light chain activated protein C have been examined. With the cations Cs+, K+, Li+, and Tl+, a single cation site, or class of sites, has been found to be responsible for the stimulation observed, with kinetic Ka values of 98-110, 180-210, 300-310, and 14-16 mM, respectively. The mechanism proposed for participation of these cations in the enzyme reaction involves an ordered addition, with the binding of cation preceding the binding of the amide substrate. On the other hand, the kinetic properties of this same activation by Na+ are consistent with either two cation sites, or classes of sites, of importance. Once again, however, the mechanism of the reaction appears to be of the ordered type, with cation binding occurring prior to substrate binding.     

Effects of Monovalent and Divalent Cations on 3-(+)[125I]Iododizocilpine Binding to the N-Methyl-d-Aspartate Receptor of Rat Brain Membranes

We have investigated the binding of 3-[125I]iododizocilpine ([125I]iodo-MK-801) to the N-methyl-D-aspartate (NMDA) receptor in well-washed rat brain membranes. [125I]Iododizocipline binding was displaced by the following: dizocilpine greater than thienylphencyclidine greater than phencyclidine greater than ketamine. Binding of [125I]iododizocilpine was enhanced by glutamate, glycine, and spermidine, whose actions could be reversed by CGS-19755, 7-chlorokynurenate, and arcaine, respectively. [125I]Iododizocilpine binding was also enhanced by a number of divalent cations, including Ba2+, Ca2+, Mg2+, Mn2+, and Sr2+, and several monovalent cations, including Na+ and K+. These cations enhanced [125I]iododizocilpine binding by an action at the polyamine site. In addition, the inhibitory effects associated with high concentrations of these cations was markedly reduced compared to those found in previous studies with [3H]dizocilpine. Analysis of the ability of spermidine, Mg2+, and Sr2+ to alter the inhibition of [125I]iododizocilpine by arcaine gave pA2 values of 5.41, 4.47, and 4.93, corresponding to EC50 concentrations of 3.9, 34.7, and 12.0 microM, respectively, suggesting that physiological concentrations of Mg2+ may occupy the polyamine site. These results demonstrate that [125I]iododizocilpine is a useful probe for the NMDA receptor. Moreover, its high specific activity and relative insensitivity to the inhibitory actions of divalent cations should make [125I]iododizocilpine a valuable ligand for the study of NMDA receptors in intact cellular systems.     

Regulation by divalent cations of 3H-baclofen binding to GABAB sites in rat cerebellar membranes

When investigating the effects of divalent cations (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+ and Ni2+) on 3H-baclofen binding to rat cerebellar synaptic membranes, we found that the specific binding of 3H-baclofen was not only dependent on divalent cations, but was increased dose-dependently in the presence of these cations. The effects were in the following order of potency: Mn2+ congruent to Ni2+ greater than Mg2+ greater than Ca2+ greater than Sr2+ greater than Ba2+. Scatchard analysis of the binding data revealed a single component of the binding sites in the presence of 2.5 mM MgCl2, 2.5 mM CaCl2 or 0.3 mM MnCl2 whereas two components appeared in the presence of 2.5 mM MnCl2 or 1 mM NiCl2. In the former, divalent cations altered the apparent affinity (Kd) without affecting density of the binding sites (Bmax). In the latter, the high-affinity sites showed a higher affinity and lower density of the binding sites than did the single component of the former. As the maximal effects of four cations (Mg2+, Ca2+, Mn2+ and Ni2+) were not additive, there are probably common sites of action of these divalent cations. Among the ligands for GABAB sites, the affinity for (-), (+) and (+/-) baclofen, GABA and beta-phenyl GABA increased 2-6 fold in the presence of 2.5 mM MnCl2, in comparison with that in HEPES-buffered Krebs solution (containing 2.5 mM CaCl2 and 1.2 mM MgSO4), whereas that for muscimol was decreased to one-fifth. Thus, the affinity of GABAB sites for its ligands is probably regulated by divalent cations, through common sites of action.     

The effects of calcium ions on double helical forms of gramicidin

The effects of binding calcium ions to the double helical forms of gramicidin present in methanol solution were examined using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. It was found that calcium ions principally alter the relative composition of the equilibrium mixture of gramicidin conformers present in the solvent. In the absence of calcium, both parallel and antiparallel double helices are present. However, the addition of small amounts of Ca²⁺ shifts the equilibrium towards the left-handed parallel double helical form. This conformational change prevents monovalent cations (caesiums) from binding to the gramicidin double helix, and even converts the shorter, wider anti-parallel double helical form normally produced in the presence of caesium into the longer, narrower parallel double helical form. Furthermore, a temperature study showed that calcium ions tend to stabilize this form relative to the ion-free forms. The conformation of gramicidin is further changed, becoming a disordered structure, when the concentration of Ca²⁺ is raised. Thus, the binding of divalent calcium ions has a number of dramatic effects on the conformations of gramicidin present in solution.     

Functional role and affinity of inorganic cations in stabilizing the tetrameric structure of the KcsA K+ channel

Crystal structures of the tetrameric KcsA K+ channel reveal seven distinct binding sites for K+ ions within the central pore formed at the fourfold rotational symmetry axis. Coordination of an individual K+ ion by eight protein oxygen atoms within the selectivity filter suggests that ion-subunit bridging by cation-oxygen interactions contributes to structural stability of the tetramer. To test this hypothesis, we examined the effect of inorganic cations on the temperature dependence of the KcsA tetramer as monitored by SDS-PAGE. Inorganic cations known to permeate or strongly block K+ channels (K+, Rb+, Cs+, Tl+, NH4+, Ba2+, and Sr2+) confer tetramer stability at higher temperatures (T0.5 range = 87 degrees C to >99 degrees C) than impermeant cations and weak blockers (Li+, Na+, Tris+, choline+; T0.5 range = 59 degrees C to 77 degrees C). Titration of K+, Ba2+, and other stabilizing cations protects against rapid loss of KcsA tetramer observed in 100 mM choline Cl at 90 degrees C. Tetramer protection titrations of K+, Rb+, Cs+, Tl+, and NH4+ at 85 degrees C or 90 degrees C exhibit apparent Hill coefficients (N) ranging from 1.7 to 3.3 and affinity constants (K0.5) ranging from 1.1 to 9.6 mM. Ba2+ and Sr2+ titrations exhibit apparent one-site behavior (N congruent with 1) with K0.5 values of 210 nM and 11 microM, respectively. At 95 degrees C in the presence of 5 mM K+, titration of Li+ or Na+ destabilizes the tetramer with K0.5 values of 57 mM and 109 mM, respectively. We conclude that specific binding interactions of inorganic cations with the selectivity filter are an important determinant of tetramer stability of KscA.