Altered Ionic Selectivity of the Sodium Channel Revealed by Cysteine Mutations within the Pore

To explore the role of pore-lining amino acids in Na⁺ channel ion-selectivity, pore residues were  replaced serially with cysteine in cloned rat skeletal muscle Na⁺ channels. Ionic selectivity was determined by measuring permeability and ionic current ratios of whole-cell currents in Xenopus oocytes. The rSkM1 channels displayed an ionic selectivity sequence Na⁺>Li⁺>NH₄ ⁺>>K⁺>>Cs⁺ and were impermeable to divalent cations.  Replacement of residues in domain IV showed significantly enhanced current and permeability ratios of NH₄ ⁺ and K⁺, and negative shifts in the reversal potentials recorded in the presence of external Na⁺ solutions when compared to cysteine mutants in domains I, II, and III (except K1237C). Mutants in domain IV showed altered selectivity sequences: W1531C (NH₄ ⁺>K⁺>Na⁺≥Li⁺≈Cs⁺), D1532C, and G1533C (Na⁺>Li⁺≥NH₄ ⁺>K⁺>Cs⁺). Conservative replacement of the aromatic residue in domain IV (W1531) with phenylalanine or tyrosine retained Na⁺ selectivity of the channel while the alanine mutant (W1531A) reduced ion selectivity. A single mutation within the third pore forming region (K1237C) dramatically altered the selectivity sequence of the rSkM1 channel (NH₄ ⁺>K⁺>Na⁺≥Li⁺≈Cs⁺) and was permeable to divalent cations having the selectivity sequence Ca²⁺≥Sr²⁺>Mg²⁺>Ba²⁺. Sulfhydryl modification of K1237C, W1531C or D1532C with methanethiosulfonate derivatives that introduce a positively charged ammonium group, large trimethylammonium moiety, or a negatively charged sulfonate group within the pore was ineffective in restoring Na⁺ selectivity to these channels. Selectivity of D1532C mutants could be largely restored by increasing extracellular pH suggesting altering the ionized state at this position influences selectivity. These data suggest that K1237 in domain III and W1531, D1532, and G1533 in domain IV play a critical role in determining the ionic selectivity of the Na⁺ channel.     

Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels

Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K⁺, Na⁺, Cs⁺ and Li⁺; organic cations: TMA and TEA, and divalents: Ba²⁺, Ca²⁺, Mg²⁺ and Mn²⁺. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs⁺, Na⁺ and K⁺ had chord conductances in the range of 35–55 pS with the exception of Li⁺, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K⁺, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba²⁺ and 15 pS for Ca²⁺ at -80 mV and 10 pS for Mg²⁺ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K⁺ currents much like the divalents. As expected, the channel K⁺ conductance increased with K⁺ concentration saturating at ~ 45 pS and the K_D of K⁺ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.     

Adenosine triphosphatase from soybean callus and root cells

The ATPase activity of a membrane fraction from soybean (Glycine max L.) root and callus cells, presumed to be enriched in plasma membrane, has been characterized with respect to ion stimulation, pH requirement, and nucleotide specificity. The enzyme from both sources was activated by divalent cations (Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺ > Sr²⁺) and further stimulated by monovalent salts. Preparations from root cells were stimulated by monovalent ions according to the sequence: K⁺ > Rb⁺ > Choline⁺ > Na⁺ > Li⁺ > NH₄⁺ > Cs⁺ > tris⁺. Membrane preparations from callus cells showed similar stimulatory patterns except for a slight preference for Na⁺ over K⁺. No synergism between K⁺ and Na⁺ was found with preparations from either cell source.     

Ion conduction and selectivity in acid-sensing ion channel 1

The ability of acid-sensing ion channels (ASICs) to discriminate among cations was assessed based on changes in conductance and reversal potential with ion substitution. Human ASIC1a was expressed in Xenopus laevis oocytes, and acid-induced currents were measured using two-electrode voltage clamp. Replacement of extracellular Na⁺ with Li⁺, K⁺, Rb⁺, or Cs⁺ altered inward conductance and shifted the reversal potentials consistent with a selectivity sequence of Li ∼ Na > K > Rb > Cs. Permeability decreased more rapidly than conductance as a function of atomic size, with P_K/P_Na = 0.1 and G_K/G_Na = 0.7 and P_Rb/P_Na = 0.03 and G_Rb/G_Na = 0.3. Stimulation of Cl⁻ currents when Na⁺ was replaced with Ca²⁺, Sr²⁺, or Ba²⁺ indicated a finite permeability to divalent cations. Inward conductance increased with extracellular Na⁺ in a hyperbolic manner, consistent with an apparent affinity (K_m) for Na⁺ conduction of 25 mM. Nitrogen-containing cations, including NH₄⁺, NH₃OH⁺, and guanidinium, were also permeant. In addition to passing through the channels, guanidinium blocked Na⁺ currents, implying competition for a site within the pore. The role of negative charges in an external vestibule of the pore was evaluated using the point mutation D434N. The mutant channel had a decreased single-channel conductance, measured in excised outside-out patches, and a macroscopic slope conductance that increased with hyperpolarization. It had a weakened interaction with Na⁺ (K_m = 72 mM) and a selectivity that was shifted toward larger atomic sizes. We conclude that the selectivity of ASIC1 is based at least in part on interactions with binding sites both within and internal to the outer vestibule.     

Structural basis of sodium–potassium exchange of a human telomeric DNA quadruplex without topological conversion

Understanding the mechanism of Na⁺/K⁺-dependent spectral conversion of human telomeric G-quadruplex (G4) sequences has been limited not only because of the structural polymorphism but also the lack of sufficient structural information at different stages along the conversion process for one given oligonucleotide. In this work, we have determined the topology of the Na⁺ form of Tel23 G4, which is the same hybrid form as the K⁺ form of Tel23 G4 despite the distinct spectral patterns in their respective nuclear magnetic resonance (NMR) and circular dichroism spectra. The spectral difference, particularly the well-resolved imino proton NMR signals, allows us to monitor the structural conversion from Na⁺ form to K⁺ form during Na⁺/K⁺ exchange. Time-resolved NMR experiments of hydrogen–deuterium exchange and hybridization clearly exclude involvement of the global unfolding for the fast Na⁺/K⁺ spectral conversion. In addition, the K⁺ titration monitored by NMR reveals that the Na⁺/K⁺ exchange in Tel23 G4 is a two-step process. The addition of K⁺ significantly stabilizes the unfolding kinetics of Tel23 G4. These results offer a possible explanation of rapid spectral conversion of Na⁺/K⁺ exchange and insight into the mechanism of Na⁺/K⁺ structural conversion in human telomeric G4s.     

Ionic interactions of Ba2+ blockades in the MthK K+ channel

The movement and interaction of multiple ions passing through in single file underlie various fundamental K⁺ channel properties, from the effective conduction of K⁺ ions to channel blockade by Ba²⁺ ions. In this study, we used single-channel electrophysiology and x-ray crystallography to probe the interactions of Ba²⁺ with permeant ions within the ion conduction pathway of the MthK K⁺ channel. We found that, as typical of K⁺ channels, the MthK channel was blocked by Ba²⁺ at the internal side, and the Ba²⁺-blocking effect was enhanced by external K⁺. We also obtained crystal structures of the MthK K⁺ channel pore in both Ba²⁺–Na⁺ and Ba²⁺–K⁺ environments. In the Ba²⁺–Na⁺ environment, we found that a single Ba²⁺ ion remained bound in the selectivity filter, preferably at site 2, whereas in the Ba²⁺–K⁺ environment, Ba²⁺ ions were predominantly distributed between sites 3 and 4. These ionic configurations are remarkably consistent with the functional studies and identify a molecular basis for Ba²⁺ blockade of K⁺ channels.     

Selectivity of the Ca2+-activated and Light-dependent K+ Channels for Monovalent Cations

The ionic selectivity of the Ca²⁺-activated K⁺ channel of Aplysia neurons and of the light-dependent K⁺ channel of Pecten photoreceptors to metal and organic cations was studied. The selectivity sequence determined from reversal potential measurements is T1⁺ K⁺ > Rb⁺ > NH⁺₄ > Cs⁺ > Na⁺, Li⁺ and is identical to the sequence determined previously for voltage-dependent K⁺ channels in a variety of tissues. Our results suggest that some physical aspect of the K⁺ channel is conserved in phyllogenetically different tissues and cells.     

Na+(Li+)/Branched-chain amino acid cotransport inPseudomonas aeruginosa

Summary A transport system for branched-chain amino acids (designated as LIV-II system) inPseudomonas aeruginosa requires Na⁺ for its operation. Coupling cation for this system was identified by measuring cation movement during substrate entry using cation-selective electrodes. Uptakes of Na⁺ and Li^– were induced by the imposition of an inwardly-directed concentration gradient of leucine, isoleucine, or valine. No uptake of H^– was found, however, under the same conditions. In addition, effects of Na⁺ and Li⁺ on the kinetic property of the system were examined. At chloride salt concentration of 2.5mm, values of apparentK _m andV _max for leucine uptake were larger in the presence of Na⁺ than Li⁺. These results indicate that the LIV-II transport system is a Na⁺(Li⁺)/substrate cotransport system, although effects of Na⁺ and Li⁺ on kinetics of the system are different.     

Microsomal (Na + K)-activated ATPase from frog skin epithelium. Cation activations and some effects of inhibitors

A method is described for the extraction of microsomal ouabain-sensitive (Na⁺ + K⁺)-activated ATPase from separated frog skin epithelium. The method yields a microsomal fraction containing (Na⁺ + K⁺)-stimulated activity in the range of 30–40 nmol · mg⁻¹ · min⁻¹ at 26 °C. This portion, which is also ouabain sensitive, is about half of the total activity in media containing Mg²⁺, Na⁺ and K⁺. These preparations also contain Mg²⁺-dependent or Ca²⁺-dependent activities which are not additive and which are not significantly affected by ouabain, Na⁺, K⁺ or Li⁺.The activations of the ouabain-sensitive ATPase activity by Mg²⁺, Na⁺, and K⁺ are similar to those described in other tissues. It is found that Li⁺ does not substitute for Na⁺ as an activator but in high concentrations does produce partial activation in the presence of Na⁺ with no K⁺. These results are pertinent to the reported observations of ouabain-sensitive Li⁺ flux across frog skin. It is concluded that this flux is not apparently due to a direct activating effect of Li⁺ on the sodium pump.     

Ionic Regulation for Cytokinin-dependent Betacyanin Synthesis in Amaranthus Seedlings

Potassium ions at low concentrations stimulate cytokinin-dependent betacyanin synthesis in Amaranthus tricolor seedlings more than other alkali metal ions when tested as the chloride salts. The sequence of relative stimulation is K⁺ > Rb⁺ > (Na⁺ = Li⁺). Calcium and Mg²⁺ ions are inhibitory at concentrations > 1 millimolar when tested as chlorides. Anions also have an effect on the degree of alkali metal stimulation in the order PO₄³⁻ > NO₃⁻ > Cl⁻. The high activity of phosphate may be partly due to its chelating effect on inhibitory Ca²⁺ ions, or to effects on K⁺ uptake. A mixture of Na⁺ and K⁺ in the presence of phosphate is more effective than either cation alone. This result may be due either to effects on tyrosine transport or on the potassium uptake system. Phytochrome-dependent betacyanin synthesis shows the same stimulation by Na⁺ plus K⁺. The effect of a number of inhibitors of transport systems on betacyanin accumulation is reported. The possible role of the ionic environment of cells in their metabolic regulation is discussed, particularly in relation to cytokinin action.     

A computational study of barium blockades in the KcsA potassium channel based on multi-ion potential of mean force calculations and free energy perturbation

Electrophysiological studies have established that the permeation of Ba²⁺ ions through the KcsA K⁺-channel is impeded by the presence of K⁺ ions in the external solution, while no effect is observed for external Na⁺ ions. This Ba²⁺ “lock-in” effect suggests that at least one of the external binding sites of the KcsA channel is thermodynamically selective for K⁺. We used molecular dynamics simulations to interpret these lock-in experiments in the context of the crystallographic structure of KcsA. Assuming that the Ba²⁺ is bound in site S₂ in the dominant blocked state, we examine the conditions that could impede its translocation and cause the observed “lock-in” effect. Although the binding of a K⁺ ion to site S₁ when site S₂ is occupied by Ba²⁺ is prohibitively high in energy (>10 kcal/mol), binding to site S₀ appears to be more plausible (ΔG > 4 kcal/mol). The 2D potential of mean force (PMF) for the simultaneous translocation of Ba²⁺ from site S₂ to site S₁ and of a K⁺ ion on the extracellular side shows a barrier that is consistent with the concept of external lock-in. The barrier opposing the movement of Ba²⁺ is very high when a cation is in site S₀, and considerably smaller when the site is unoccupied. Furthermore, free energy perturbation calculations show that site S₀ is selective for K⁺ by 1.8 kcal/mol when S₂ is occupied by Ba²⁺. However, the same site S₀ is nonselective when site S₂ is occupied by K⁺, which shows that the presence of Ba²⁺ affects the selectivity of the pore. A theoretical framework within classical rate theory is presented to incorporate the concentration dependence of the external ions on the lock-in effect.     

Conformational switch of insulin-binding aptamer into G-quadruplex induced by K+ and Na+: an experimental and theoretical approach

Guanine-rich sequences can form the G-quadruplex structure in the presence of specific metal ions. Here, circular dichroism, UV–vis absorption, fluorescence, and molecular dynamics simulation studies revealed that insulin-binding aptamer (IBA) could form an intramolecular G-quadruplex structure after binding K⁺. Circular dichroism (CD) spectra demonstrated that IBA could fold into a parallel G-quadruplex with a strong positive peak at 263?nm. Analysis of equilibrium titration data revealed that cation binding was cooperative with the Hill coefficient of 2.01 in K⁺ and 1.90 in Na⁺. Thermal denaturation assays indicated that K⁺-induced G-quadruplex is more stable than Na⁺-induced structure. Folding of IBA into G-quadruplex leading to the contact quenching occurs as a result of the formation of a nonfluorescent complex between donor and acceptor. Based on fluorescence quenching of IBA folding, a potassium-sensing aptasensor in the range of 0–1.4?mM was proposed. Since the quenching process was predominantly static, the binding constant and the number of binding sites were determined. In this research, based on the experimental data, the initial model of IBA G-quadruplex was constructed by molecular modeling method. The modeling structure of IBA is an intramolecular parallel-strand quadruplex conformation with two guanine tetrads. The extended molecular dynamics simulation for the model indicated that the G-quadruplex maintains its structure very well in aqueous solution in presence of K⁺ in the central cavity. In contrast, it was demonstrated that the G-quadruplex structure of model in the water collapses in absence of this cation.     

BIOELECTRIC POTENTIALS IN VALONIA : II. EFFECTS OF ARTIFICIAL SEA WATERS CONTAINING LiCl,CsCl, RbCl,OR NH4Cl

In their influence on the P.D. across the protoplasm of Valonia macrophysa, Kütz., Li⁺ and Cs⁺ resemble Na⁺, while Rb⁺ and NH₄ ⁺ resemble K⁺. The apparent mobilities of the ions in the external surface layer of Valonia protoplasm increase in the order: Cs⁺, Na⁺, Li⁺ < Cl^- < Rb⁺ < K⁺ < NH₄ ⁺.     

A fluorescent aptasensor for potassium ion detection-based triple-helix molecular switch

Here, a biosensor based on a quadruplex-forming aptamer for the determination of potassium ion (K⁺) is presented. The aptamer was used as a molecular recognition element; it was adjacent to two arm fragments and a dual-labeled oligonucleotide serving as a signal transduction probe (STP) that is complementary of the arm fragment sequence. In the presence of K⁺, the aptamer was displaced from the STP, which was accompanied by decreased signal. The quenching percentage of fluorescence intensity was proportional to the concentration of K⁺ in the range of 0.05 to 1.4 mM. A detection limit of 0.014 mM was achieved. Furthermore, other metal ions, such as Na⁺, Li⁺, NH₄⁺, Mg²⁺, and Ca²⁺, caused no notable interference on the detection of K⁺.     

Interaction of the Depolarization-Activated K Channel of Samanea saman with Inorganic Ions: A Patch-Clamp Study

A depolarization-activated K⁺ channel capable of carrying the large K⁺ currents that flow from shrinking cells during movements of Samanea saman leaflets has been described in the plasmalemma of Samanea motor cell protoplasts (N Moran et al [1988] Plant Physiol 88:643-648). We now characterize this channel in greater detail. It is selective for K⁺ over other monovalent ions, with the following order of relative permeability: K⁺ > Rb⁺ > Na⁺ Cs⁺ Li⁺. It is blocked by Cs⁺ and by Ba²⁺ in a voltage dependent manner, exhibiting a `long-pore' behavior, similarly to various types of K⁺ channels in animal systems. Cadmium, known for its blockage of Ca²⁺ channels in animal systems, and Gd³⁺, closely related to La³⁺, which also blocks Ca²⁺ channels in animal cells, both block K⁺ currents in Samanea in a voltage-independent manner, and without interfering with the kinetics of the currents. The suggested mechanism of block is either (a) by a direct interaction with the K⁺ channel, but external to its lumen, or, alternatively, (b) by blocking putative Ca²⁺ channels, and preventing the influx of Ca²⁺, on which the activation of the K⁺ channels may be dependent.     

Functional reconstitution of the mitochondrial Ca2+/H+ antiporter Letm1

The leucine zipper, EF hand–containing transmembrane protein 1 (Letm1) gene encodes a mitochondrial inner membrane protein, whose depletion severely perturbs mitochondrial Ca²⁺ and K⁺ homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca²⁺ fluorophore and ⁴⁵Ca²⁺-based assays, we demonstrate directly that Letm1 is a Ca²⁺ transporter, with apparent affinities of cations in the sequence of Ca²⁺ ≈ Mn²⁺ > Gd³⁺ ≈ La³⁺ > Sr²⁺ >> Ba²⁺, Mg²⁺, K⁺, Na⁺. Kinetic analysis yields a Letm1 turnover rate of 2 Ca²⁺/s and a K_m of ∼25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca²⁺/2 H⁺ antiport. Letm1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na⁺/Ca²⁺ exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H⁺-dependent Ca²⁺ transport mechanism identified in intact mitochondria.     

Block of N-type Calcium Channels in Chick Sensory Neurons by External Sodium

L-type Ca²⁺ channels select for Ca²⁺ over sodium Na⁺ by an affinity-based mechanism. The prevailing model of Ca²⁺ channel permeation describes a multi-ion pore that requires pore occupancy by at least two Ca²⁺ ions to generate a Ca²⁺ current. At [Ca²⁺] < 1 μM, Ca²⁺ channels conduct Na⁺. Due to the high affinity of the intrapore binding sites for Ca²⁺ relative to Na⁺, addition of μM concentrations of Ca²⁺ block Na⁺ conductance through the channel. There is little information, however, about the potential for interaction between Na⁺ and Ca²⁺ for the second binding site in a Ca²⁺ channel already occupied by one Ca²⁺. The two simplest possibilities, (a) that Na⁺ and Ca²⁺ compete for the second binding site or (b) that full time occupancy by one Ca²⁺ excludes Na⁺ from the pore altogether, would imply considerably different mechanisms of channel permeation. We are studying permeation mechanisms in N-type Ca²⁺ channels. Similar to L-type Ca²⁺ channels, N-type channels conduct Na⁺ well in the absence of external Ca²⁺. Addition of 10 μM Ca²⁺ inhibited Na⁺ conductance by 95%, and addition of 1 mM Mg²⁺ inhibited Na⁺ conductance by 80%. At divalent ion concentrations of 2 mM, 120 mM Na⁺ blocked both Ca²⁺ and Ba²⁺ currents. With 2 mM Ba²⁺, the IC₅₀ for block of Ba²⁺ currents by Na⁺ was 119 mM. External Li⁺ also blocked Ba²⁺ currents in a concentration-dependent manner, with an IC₅₀ of 97 mM. Na⁺ block of Ba²⁺ currents was dependent on [Ba²⁺]; increasing [Ba²⁺] progressively reduced block with an IC₅₀ of 2 mM. External Na⁺ had no effect on voltage-dependent activation or inactivation of the channel. These data suggest that at physiological concentrations, Na⁺ and Ca²⁺ compete for occupancy in a pore already occupied by a single Ca²⁺. Occupancy of the pore by Na⁺ reduced Ca²⁺ channel conductance, such that in physiological solutions, Ca²⁺ channel currents are between 50 and 70% of maximal.     

Structure of the human telomere in Na+ solution: an antiparallel (2+2) G-quadruplex scaffold reveals additional diversity

Single-stranded DNA overhangs at the ends of human telomeric repeats are capable of adopting four-stranded G-quadruplex structures, which could serve as potential anticancer targets. Out of the five reported intramolecular human telomeric G-quadruplex structures, four were formed in the presence of K⁺ ions and only one in the presence of Na⁺ ions, leading often to a perception that this structural polymorphism occurs exclusively in the presence of K⁺ but not Na⁺. Here we present the structure of a new antiparallel (2+2) G-quadruplex formed by a derivative of a 27-nt human telomeric sequence in Na⁺ solution, which comprises a novel core arrangement distinct from the known topologies. This structure complements the previously elucidated basket-type human telomeric G-quadruplex to serve as reference structures in Na⁺-containing environment. These structures, together with the coexistence of other conformations in Na⁺ solution as observed by nuclear magnetic resonance spectroscopy, establish the polymorphic nature of human telomeric repeats beyond the influence of K⁺ ions.     

Characterization of a k-stimulated adenosine triphosphatase associated with the plasma membrane of red beet

A membrane fraction enriched with a magnesium-dependent, monovalent cation-stimulated ATPase was isolated from red beet (Beta vulgaris L.) storage roots by a combination of differential centrifugation, extraction with KI, and sucrose density gradient centrifugation. This fraction was distinct from endoplasmic reticulum, Golgi, mitochondrial, and possibly tonoplast membranes as determined from an analysis of marker enzymes. The ATPase activity associated with this fraction was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was substrate specific for ATP and had a temperature optimum near 40°C. Kinetics with Mg:ATP followed a simple Michaelis-Menten relationship. However the kinetics of K⁺-stimulation were complex and suggestive of negative cooperativity. When monovalent cations were present at 2.5 millimolarity, ATPase was stimulated in the sequence K⁺ > Rb⁺ > Na⁺ > Li⁺ but when the concentration was raised to 50 millimolarity, the sequence changed to K⁺ ≥ Na⁺ ≥ Rb⁺ > Li. The activity was not synergistically stimulated by combinations of Na⁺ and K⁺. The enzyme was insensitive to NaN₃, oligomycin, ouabain, and sodium molybdate but sensitive to N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and sodium vanadate. Based on the similarity between the properties of this ATPase activity and those from other well characterized plant tissues, it has been concluded that this membrane fraction is enriched with plasma membrane vesicles.     

Conformational change of a G-quadruplex under molecular crowding conditions

Abstract

This study examined the influence of the molecular crowding condition induced by polyethylene glycol (PEG) on the G-quadruplex structure of the thrombin-binding aptamer sequence, 5′-GGGTTGGGTGTGGGTTGGG (G3), in a solution containing a sufficient concentration of mono cations (K⁺ and Na⁺). Although the G3 sequence preferably formed the antiparallel type G-quadruplex structure in a Na⁺ solution, conversion to the parallel type occurred when PEG was added. The antiparallel type was maintained at low PEG concentrations. When the PEG concentration reached 30%, the antiparallel type and parallel type coexist. At PEG concentrations above 40%, the G-quadruplex structure adopted the parallel type completely. In the presence of K⁺ ions, G3 showed a parallel conformation and remained as a parallel conformation with increasing PEG concentration. The dissociation temperature increased with increasing PEG concentration in all cases, suggesting that the G-quadruplex conformation is more stable under molecular crowding conditions.

Communicated by Ramaswamy H. Sarma