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.     

Not so crystal clear: the structure of the human telomere G-quadruplex in solution differs from that present in a crystal

The structure of human telomere DNA is of intense interest because of its role in the biology of both cancer and aging. The sequence [5′-AGGG(TTAGGG)₃] has been used as a model for telomere DNA in both NMR and X-ray crystallographic studies, the results of which show dramatically different structures. In Na⁺ solution, NMR revealed an antiparallel G-quadruplex structure that featured both diagonal and lateral TTA loops. Crystallographic studies in the presence of K⁺ revealed a flattened, propeller-shaped structure featuring a parallel-stranded G-quadruplex with symmetrical external TTA loops. We report the results of biophysical experiments in solution and computational studies that are inconsistent with the reported crystal structure, indicating that a different structure exists in K⁺ solutions. Sedimentation coefficients were determined experimentally in both Na⁺ and K⁺ solutions and were compared with values calculated using bead models for the reported NMR and crystal structures. Although the solution NMR structure accurately predicted the observed S-value in Na⁺ solution, the crystal structure predicted an S-value that differed dramatically from that experimentally observed in K⁺ solution. The environments of loop adenines were probed by quantitative fluorescence studies using strategic and systematic single-substitutions of 2-aminopurine for adenine bases. Both fluorescence intensity and quenching experiments in K⁺ yielded results at odds with quantitative predictions from the reported crystal structure. Circular dichroism and fluorescence quenching studies in the presence of the crowding agent polyethylene glycol showed dramatic changes in the quadruplex structure in K⁺ solutions, but not in Na⁺ solutions, suggesting that the crystal environment may have selected for a particular conformational form. Molecular dynamics simulations were performed to yield model structures for the K⁺ quadruplex form that are consistent with our biophysical results and with previously reported chemical modification studies. These models suggest that the biologically relevant structure of the human telomere quadruplex in K⁺ solution is not the one determined in the published crystalline state.     

Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). The formation and stabilization of DNA G-quadruplexes in the human telomeric sequence have been shown to inhibit the activity of telomerase, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. However, knowledge of the intact human telomeric G-quadruplex structure(s) formed under physiological conditions is a prerequisite for structure-based rational drug design. Here we report the folding structure of the human telomeric sequence in K⁺ solution determined by NMR. Our results demonstrate a novel, unprecedented intramolecular G-quadruplex folding topology with hybrid-type mixed parallel/antiparallel G-strands. This telomeric G-quadruplex structure contains three G-tetrads with mixed G-arrangements, which are connected consecutively with a double-chain-reversal side loop and two lateral loops, each consisting of three nucleotides TTA. This intramolecular hybrid-type telomeric G-quadruplex structure formed in K⁺ solution is distinct from those reported on the 22 nt Tel22 in Na⁺ solution and in crystalline state in the presence of K⁺, and appears to be the predominant conformation for the extended 26 nt telomeric sequence Tel26 in the presence of K⁺, regardless of the presence or absence of Na⁺. Furthermore, the addition of K⁺ readily converts the Na⁺-form conformation to the K⁺-form hybrid-type G-quadruplex. Our results explain all the reported experimental data on the human telomeric G-quadruplexes formed in the presence of K⁺, and provide important insights for understanding the polymorphism and interconversion of various G-quadruplex structures formed within the human telomeric sequence, as well as the effects of sequence and cations. This hybrid-type G-quadruplex topology suggests a straightforward pathway for the secondary structure formation with effective packing within the extended human telomeric DNA. The hybrid-type telomeric G-quadruplex is most likely to be of pharmacological relevance, and the distinct folding topology of this G-quadruplex suggests that it can be specifically targeted by G-quadruplex interactive small molecule drugs.     

Natural isoflavones regulate the quadruplex–duplex competition in human telomeric DNA

Effects of natural isoflavones on the structural competition of human telomeric G-quadruplex d[AG₃(T₂AG₃)₃] and its related Watson–Crick duplex d[AG₃(T₂AG₃)₃-(C₃TA₂)₃C₃T] are investigated by using circular dichroism (CD), ESI-MS, fluorescence quenching measurement, CD stopped-flow kinetic experiment, UV spectroscopy and molecular modeling methods. It is intriguing to find out that isoflavones can stabilize the G-quadruplex structure but destabilize its corresponding Watson–Crick duplex and this discriminated interaction is intensified by molecular crowding environments. Kinetic experiments indicate that the dissociation rate of quadruplex (k_{obs290 nm}) is decreased by 40.3% at the daidzin/DNA molar ratio of 1.0 in K⁺, whereas in Na⁺ the observed rate constant is reduced by about 12.0%. Furthermore, glycosidic daidzin significantly induces a structural transition of the polymorphic G-quadruplex into the antiparallel conformation in K⁺. This is the first report on the recognition of isoflavones with conformational polymorphism of G-quadruplex, which suggests that natural isoflavone constituents potentially exhibit distinct regulation on the structural competition of quadruplex versus duplex in human telomeric DNA.     

Single-molecule detection of folding and unfolding of the G-quadruplex aptamer in a nanopore nanocavity

Guanine-rich nucleic acids can form G-quadruplexes that are important in gene regulation, biosensor design and nano-structure construction. In this article, we report on the development of a nanopore encapsulating single-molecule method for exploring how cations regulate the folding and unfolding of the G-quadruplex formed by the thrombin-binding aptamer (TBA, GGTTGGTGTGGTTGG). The signature blocks in the nanopore revealed that the G-quadruplex formation is cation-selective. The selectivity sequence is K⁺ > NH₄⁺ ~ Ba²⁺ > Cs⁺ ~ Na⁺ > Li⁺, and G-quadruplex was not detected in Mg²⁺ and Ca²⁺. Ba²⁺ can form a long-lived G-quadruplex with TBA. However, the capability is affected by the cation–DNA interaction. The cation-selective formation of the G-quadruplex is correlated with the G-quadruplex volume, which varies with cation species. The high formation capability of the K⁺-induced G-quadruplex is contributed largely by the slow unfolding reaction. Although the Na⁺- and Li⁺-quadruplexes feature similar equilibrium properties, they undergo radically different pathways. The Na⁺-quadruplex folds and unfolds most rapidly, while the Li⁺-quadruplex performs both reactions at the slowest rates. Understanding these ion-regulated properties of oligonucleotides is beneficial for constructing fine-tuned biosensors and nano-structures. The methodology in this work can be used for studying other quadruplexes and protein–aptamer interactions.     

Direct NMR detection of the "invisible" alkali metal cations tightly bound to G-quadruplex structures

We report the first direct solution NMR detection of the alkali metal cations (²³Na⁺, ³⁹K⁺, and ⁸⁷Rb⁺) residing inside G-quadruplex channel structures formed by guanosine 5′-monophosphate and a DNA oligomer, d(TG₄T). In solution, these channel alkali metal cations are tightly bound to the G-quadruplex structure and have been considered to be “invisible” to NMR spectroscopy for many years. Our finding that it is possible to directly observe these alkali metal cations by NMR spectroscopy provides a new tool for studying cation binding affinity and dynamics in G-quadruplex DNA.     

Similarities and differences in interaction of K+ and Na+ with condensed ordered DNA. A molecular dynamics computer simulation study

Four 20 ns molecular dynamics simulations have been performed with two counterions, K⁺ or Na⁺, at two water contents, 15 or 20 H₂O per nucleotide. A hexagonal simulation cell comprised of three identical DNA decamers [d(5′-ATGCAGTCAG) × d(5′-TGACTGCATC)] with periodic boundary condition along the DNA helix was used. The simulation setup mimics the DNA state in oriented DNA fibers or in crystals of DNA oligomers. Variation of counterion nature and water content do not alter averaged DNA structure. K⁺ and Na⁺ binding to DNA are different. K⁺ binds to the electronegative sites of DNA bases in the major and the minor grooves, while Na⁺ interacts preferentially with the phosphate groups. Increase of water causes a shift of both K⁺ and Na⁺ from the first hydration shell of O1P/O2P and of the DNA bases in the minor groove with lesser influence for the cation binding to the bases in the major groove. Mobility of both water and cations in the K–DNA systems is faster than in the Na–DNA systems: Na⁺ organizes and immobilizes water structure around itself and near DNA while for K⁺ water is less organized and more dynamic.     

Studies on the interaction between vincamine and human serum albumin: a spectroscopic approach

The interaction between vincamine (VCM) and human serum albumin (HSA) has been studied using a fluorescence quenching technique in combination with UV/vis absorption spectroscopy, Fourier transform infrared (FT–IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling under conditions similar to human physiological conditions. VCM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding constants were calculated from the fluorescence data. Thermodynamic analysis by Van't Hoff equation revealed enthalpy change (ΔH) and entropy change (ΔS) were ?4.57 kJ/mol and 76.26 J/mol/K, respectively, which indicated that the binding process was spontaneous and the hydrophobic interaction was the predominant force. The distance r between the donor (HSA) and acceptor (VCM) was obtained according to the Förster's theory of non‐radiative energy transfer and found to be 4.41 nm. Metal ions, viz., Na⁺, K⁺, Li⁺, Ni²⁺, Ca²⁺, Zn²⁺ and Al³⁺ were found to influence binding of the drug to protein. The 3D fluorescence, FT–IR and CD spectral results revealed changes in the secondary structure of the protein upon interaction with VCM. Furthermore, molecular modeling indicated that VCM could bind to the subdomain IIA (site I) of HSA. Copyright © 2013 John Wiley & Sons, Ltd.     

Human telomeric G-quadruplex formed from duplex under near physiological conditions: Spectroscopic evidence and kinetics

The structural interconversion between the G-quadruplex and duplex in vivo is an important subject. In the present study, we used human telomeric DNA duplex composed of GGG(TTAGGG)₃/CCC(TAACCC)₃ as a model system to investigate its properties under near physiological conditions by spectroscopic methods. Circular dichroism and fluorescence spectra demonstrated that G-quadruplex structure can be formed from duplex at near physiological pH (pH 7.4), salt concentration (150 mM K⁺), and temperature (37 °C) in the presence of molecular crowding agent PEG (400 g/l), whereas the G-quadruplex structure cannot be formed at 25 °C in buffer containing 150 mM K⁺ in the presence of PEG. It is found that the formation rate of G-quadruplex structure depends on the temperature and the concentrations of both PEG and K⁺. This work suggests that human telomeric G-quadruplex structure may be potentially formed from Watson–Crick duplex in vivo.     

Na+-K+ transport in roots under salt stress

Salinity causes billion dollar losses in annual crop production. So far, the main avenue in breeding crops for salt tolerance has been to reduce Na⁺ uptake and transport from roots to shoots. Recently we have demonstrated that retention of cytosolic K⁺ could be considered as another key factor in conferring salt tolerance in plants. A subsequent study has shown that Na⁺-induced K⁺ efflux in barley root epidermis occurs primarily via outward rectifying K⁺ channels (KORC). Surprisingly, expression of KORC was similar in salt- tolerant and sensitive genotypes. However, the former were able to better oppose Na⁺-induced depolarization via enhanced activity of plasma membrane H⁺-ATPase (thus minimizing K⁺ leak from the cytosol). In addition to highly K⁺-selective KORC channels, activities of several types of non-selective cation channels were detected at depolarizing potentials. Here we show that the expression of one of them, NORC, was significantly lower in salt-tolerant genotypes. As NORC is capable of mediating K⁺ efflux coupled to Na⁺ influx, we suggest that the restriction of its activity could be beneficial for plants under salt stress.Key words: salinity tolerance, barley, ion flux, K⁺ homeostasis, KOR, non-selective channels, patch-clamp     

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     

Control of ion selectivity in LeuT: two Na+ binding sites with two different mechanisms

The x-ray structure of LeuT, a bacterial homologue of Na⁺/Cl⁻-dependent neurotransmitter transporters, provides a great opportunity to better understand the molecular basis of monovalent cation selectivity in ion-coupled transporters. LeuT possesses two ion binding sites, NA1 and NA2, which are highly selective for Na⁺. Extensive all-atom free-energy molecular dynamics simulations of LeuT embedded in an explicit membrane are performed at different temperatures and various occupancy states of the binding sites to dissect the molecular mechanism of ion selectivity. The results show that the two binding sites display robust selectivity for Na⁺ over K⁺ or Li⁺, the competing ions of most similar radii. Of particular interest, the mechanism primarily responsible for selectivity for each of the two binding sites appears to be different. In NA1, selectivity for Na⁺ over K⁺ arises predominantly from the strong electrostatic field arising from the negatively charged carboxylate group of the leucine substrate coordinating the ion directly. In NA2, which comprises only neutral ligands, selectivity for Na⁺ is enforced by the local structural restraints arising from the hydrogen-bonding network and the covalent connectivity of the polypeptide chain surrounding the ion according to a “snug-fit” mechanism.     

(Na+,K+)-ATPase of mammalian brain: Effects of temperature on cation and ATP interactions regulating phosphatase activity

The effects of temperature on interactions between univalent cations or ATP and the p-nitrophenylphosphatase activity associated with brain (Na⁺,K⁺)-ATPase were examined. The apparent affinity for K⁺ activation under conditions favoring the moderate affinity site was temperature dependent, increasing with decreasing temperature. A comparison of univalent cations showed that the negative apparent ΔH and ΔS for cation binding increased with increasing apparent cation affinity. In contrast to the case with the moderate affinity sites, apparent affinity for the high affinity K⁺ site was independent of temperature. As temperature decreased, properties of moderate affinity site binding approached those of the high affinity site. The temperature dependence of ATP inhibition was opposite to that for K⁺ activation, with positive apparent ΔH and ΔS. The apparent ΔH and ΔS for cation binding approached those for the overall conformational change to K⁺-sensitive enzyme as cation affinity increased. These data suggest that E2, the K⁺-sensitive form of (Na⁺,K⁺)-ATPase, is stabilized by forces that require a decrease in entropy, explaining the predominant existence of E1 at physiologic temperatures. A conformational change leading to stabilization of E2 at higher temperatures can be produced by binding of univalent cations to a moderate affinity, presumably intracellular, site. This effect is counteracted by ATP. ATP also appears to alter the selectivity of this site to favor Na⁺ over K⁺ binding.     

Human telomere d[(TTAGGG)4] undergoes a conformational transition to the Na+-form upon binding with sanguinarine in presence of K+

Guanine-rich telomeric sequences fold into G-quadruplex conformation and are known to bind a variety of ligands including potential drug candidates. By means of CD spectroscopy and fluorescence lifetime measurements we demonstrate that putative anticancer therapeutic sanguinarine (SGR) exhibits two distinct interactions with human telomere d[(TTAGGG)₄] (H24) in presence of K⁺. Up to about 1:2 M ratio of H24:SGR (10 μM H24), two molecules of SGR bind H24. Above this molar ratio, SGR induces a conformational transition in H24 from the K⁺-form to the Na⁺-form. The demonstration of SGR-induced conformational transition in a G-quadruplex formed by a human telomeric sequence could provide new insights into interaction of drugs with quadruplex DNA structure.     

Interaction of [Ru(bpy)2(dppz)] with human telomeric DNA: Preferential binding to G-quadruplexes over i-motif

Inspired by the enormous importance attributed to the structure and function of human telomeric DNA, we focus our attention on the interaction of [Ru(bpy)₂(dppz)]²⁺ with the guanine-rich single-strand oligomer 5′-AGGGTTAGGGTTAGGGTTAGGG-3′ (22AG) and the complementary cytosine-rich strand (22CT). In Na⁺ buffer, 22AG may adopt an antiparallel basket quadruplex, whereas, it favours a mixed parallel/antiparallel structure in K⁺ buffer. 22CT may self-associate at acidic pH into an i-motif. In this paper, the interaction between [Ru(bpy)₂(dppz)]²⁺ and each unusual DNA was evaluated. It was interesting that [Ru(bpy)₂(dppz)]²⁺ could promote the human telomeric repeat 22AG to fold into intramolecular antiparallel G-quadruplex without any other cations. What's more, [Ru(bpy)₂(dppz)]²⁺ was found to have a strong preference for binding to G-quadruplexes that were induced through either Na⁺ or K⁺, while weak binding to i-motif was observed. The results also indicated that [Ru(bpy)₂(dppz)]²⁺ could serve as a prominent molecular “light switch” for both G-quadruplexes, revealing a potential application of the title complex in luminescent signaling of G-quadruplex DNA.     

Structure of the Hybrid-2 type intramolecular human telomeric G-quadruplex in K+ solution: insights into structure polymorphism of the human telomeric sequence

Formation of the G-quadruplex in the human telomeric sequence can inhibit the activity of telomerase, thus the intramolecular telomeric G-quadruplexes have been considered as an attractive anticancer target. Information of intramolecular telomeric G-quadruplex structures formed under physiological conditions is important for structure-based drug design. Here, we report the first structure of the major intramolecular G-quadruplex formed in a native, non-modified human telomeric sequence in K⁺ solution. This is a hybrid-type mixed parallel/antiparallel-G-stranded G-quadruplex, one end of which is covered by a novel T:A:T triple capping structure. This structure (Hybrid-2) and the previously reported Hybrid-1 structure differ in their loop arrangements, strand orientations and capping structures. The distinct capping structures appear to be crucial for the favored formation of the specific hybrid-type intramolecular telomeric G-quadruplexes, and may provide specific binding sites for drug targeting. Our study also shows that while the hybrid-type G-quadruplexes appear to be the major conformations in K⁺ solution, human telomeric sequences are always in equilibrium between Hybrid-1 and Hybrid-2 structures, which is largely determined by the 3′-flanking sequence. Furthermore, both hybrid-type G-quadruplexes suggest a straightforward means for multimer formation with effective packing in the human telomeric sequence and provide important implications for drug targeting of G-quadruplexes in human telomeres.     

Residues important for K+ ion transport in the K+-dependent Na+-Ca2+ exchanger (NCKX2)

K⁺-dependent Na⁺-Ca²⁺ exchangers (NCKXs) play an important role in Ca²⁺ homeostasis in many tissues. NCKX proteins are bi-directional plasma membrane Ca²⁺-transporters which utilize the inward Na⁺ and outward K⁺ gradients to move Ca²⁺ ions into and out of the cytosol (4Na⁺:1Ca²⁺ + 1 K⁺). In this study, we carried out scanning mutagenesis of all the residues of the highly conserved α-1 and α-2 repeats of NCKX2 to identify residues important for K⁺ transport. These structural elements are thought to be critical for cation transport. Using fluorescent intracellular Ca²⁺-indicating dyes, we measured the K⁺ dependence of transport carried out by wildtype or mutant NCKX2 proteins expressed in HEK293 cells and analyzed shifts in the apparent binding affinity (K_m) of mutant proteins in comparison with the wildtype exchanger. Of the 93 residue substitutions tested, 34 were found to show a significant shift in the external K⁺ ion dependence of which 16 showed an increased affinity to K⁺ ions and 18 showed a decreased affinity and hence are believed to be important for K⁺ ion binding and transport. We also identified 8 residue substitutions that resulted in a partial loss of K⁺ dependence. Our biochemical data provide strong support for the cation binding sites identified in a homology model of NCKX2 based on crystal structures reported for distantly related archaeal Na⁺-Ca²⁺ exchanger NCX_Mj. In addition, we compare our results here with our previous studies that report on residues important for Ca²⁺ and Na⁺ binding. Supported by CIHR MOP-81327.     

Apparent inhibition of Na+/H+ exchange by amiloride and harmaline in acridine orange studies

Amiloride and harmaline were tested as inhibitors of proton movements in brush-border membrane vesicles from rat kidney cortex. Transmembrane pH differences were visualized using acridine orange. Fluorescence quenching due to Na⁺ gradient-driven intravesicular acidification was inhibited by amiloride and harmaline. However, a similar inhibition was observed for the Na⁺ gradient-driven electrogenic proton movements in the presence of gramicidin. Moreover, amiloride and harmaline decreased the fluorescence signal of electrogenic proton movements driven by a K⁺ gradient in the presence of valinomycin. The degree of inhibition of intravesicular acidification by both drugs was concentration dependent. Half-maximal inhibition (I₅₀) of Na⁺/H⁺ exchange and K⁺ gradient-driven proton movements occurred at 0.21 and 0.6 amiloride, respectively. The I₅₀ for harmaline was 0.21 mM in both cases. Amiloride also decreased the initial quenching of acridine orange fluorescence due to a preset pH gradient without affecting the rate of dissipation of the pH gradient. This effect was independent of the buffer capacity. In contrast, harmaline seemed to dissipate pH gradient in the same way as a permeant buffer. Amiloride and harmaline led to a concentration-dependent fluorescence decrease even in aqueous solution. The results suggest an interaction of amiloride and harmaline with acridine orange which overlaps a possible specific inhibition of Na⁺/H⁺ exchange by these drugs.     

Na+K+-ATPase Activity and K+ Channels Differently Contribute to Vascular Relaxation in Male and Female Rats

Gender associated differences in vascular reactivity regulation might contribute to the low incidence of cardiovascular disease in women. Cardiovascular protection is suggested to depend on female sex hormones’ effects on endothelial function and vascular tone regulation. We tested the hypothesis that potassium (K⁺) channels and Na⁺K⁺-ATPase may be involved in the gender-based vascular reactivity differences. Aortic rings from female and male rats were used to examine the involvement of K⁺ channels and Na⁺K⁺-ATPase in vascular reactivity. Acetylcholine (ACh)-induced relaxation was analyzed in the presence of L-NAME (100 µM) and the following K⁺ channels blockers: tetraethylammonium (TEA, 2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 µM) and charybdotoxin (ChTX, 0.1 µM). The ACh-induced relaxation sensitivity was greater in the female group. After incubation with 4-AP the ACh-dependent relaxation was reduced in both groups. However, the dAUC was greater in males, suggesting that the voltage-dependent K⁺ channel (K_v) participates more in males. Inhibition of the three types of Ca²⁺-activated K⁺ channels induced a greater reduction in R_max in females than in males. The functional activity of the Na⁺K⁺-ATPase was evaluated by KCl-induced relaxation after L-NAME and OUAincubation. OUA reduced K⁺-induced relaxation in female and male groups, however, it was greater in males, suggesting a greater Na⁺K⁺-ATPase functional activity. L-NAME reduced K⁺-induced relaxation only in the female group, suggesting that nitric oxide (NO) participates more in their functional Na⁺K⁺-ATPase activity. These results suggest that the K⁺ channels involved in the gender-based vascular relaxation differences are the large conductance Ca²⁺-activated K⁺ channels (BK_Ca) in females and K_v in males and in the K⁺-induced relaxation and the Na⁺K⁺-ATPase vascular functional activity is greater in males.     

Proton-pumping adenosine triphosphatase in membrane vesicles of tobacco callus: Sensitivity to vanadate and K+

H⁺-pumping adenosinetriphosphatases (ATPases, EC 3.6.1.3) were demonstrated in sealed microsomal vesicles of tobacco callus. Quinacrine fluorescence quenching was induced specifically by MgATP and stimulated by EGTA and Cl^?. Fluorescence quenching reflected a relative measure of pH gradient formation (inside acid), as it could be reversed by gramicidin (an H⁺/cation conductor) or 10 mM NH₄Cl (an uncoupler). H⁺ pumping was inhibited by tributyltin (an ATPase inhibitor) and sodium vanadate, but it was insensitive to oligomycin or fusicoccin. The vanadate concentration required to inhibit pH gradient formation was similar to that needed to inhibit KCl-stimulated Mg²⁺-ATPase activity and generation of a membrane potential (measured by ATP-dependent ³⁵SCN^? uptake). About 45% of all three activities (ATPase, pH gradient, membrane potential generation) were vanadate-insensitive, supporting the idea that non-mitochondrial membranes of plants have at least two types of electrogenic H⁺ pump.A vanadate-insensitive, H⁺-pumping ATPase previously shown by methylamine accumulation was characterized to be anion-sensitive and possibly enriched in vacuolar membranes (Churchill, K.A. and Sze, H. (1983) Plant Physiol. 71, 610–617). Yet, pH gradient formation determined by quinacrine fluorescence quenching was decreased by monovalent cations with a sequence K⁺, Rb⁺, Na⁺ > Cs⁺,Li⁺> choline, bisTris-propane. Since K⁺ stimulated ATPase activity more than Bistris-propane, K⁺ appeared to collapse formation of the pH gradient by an H⁺/K⁺ countertransport. The sensitivity to vanadate and K⁺ provides evidence that the plasma-membrane ATPase is an electrogenic H⁺ pump.