Acylspermidine derivatives isolated from a soft coral,Sinularia sp,inhibit plant vacuolar H(+)-pyrophosphatase

H(+)-pyrophosphatase (H(+)-PPase), which pumps H(+) across membranes coupled with PP(i) hydrolysis, is found in most plants, and some parasitic protists, eubacteria and archaebacteria. We assayed a number of extracts derived from 145 marine invertebrates as to their inhibitory effect on plant vacuolar H(+)-PPase. Acylspermidine derivatives [RCONH(CH(2))(3)N(CH(3))(CH(2))(4)N(CH(3))(2)] from a soft coral (Sinularia sp.) inhibited the PPi-hydrolysis activity of purified H(+)-PPase and the PP(i)-dependent H(+) pump activity (half inhibition concentration, 1 micro M) of vacuolar membranes of mung bean. The apparent K(i) was determined to be 0.9 micro M. Acylspermidines did not affect the activity of vacuolar H(+)-ATPase, plasma membrane H(+)-ATPase, mitochondrial ATPase or cytosolic PPase. Acylspermidines inhibited the acidification of vacuoles in protoplasts, as found on monitoring by the acridine orange fluorescent method. These results indicate that acylspermidine derivatives represent new inhibitors of H(+)-PPase with relatively high specificity.     

A lysine residue involved in the inhibition of vacuolar H(+)-pyrophosphatase by fluorescein 5'-isothiocyanate

Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a central role in the electrogenic translocation of protons from cytosol to the vacuole lumen at the expense of PP(i) hydrolysis. A fluorescent probe, fluorescein 5'-isothiocyanate (FITC), was used to modify a lysine residue of vacuolar H(+)-PPase. The enzymatic activity and its associated H(+) translocation of vacuolar H(+)-PPase were markedly decreased by FITC in a concentration-dependent manner. The inhibition of enzymatic activity followed pseudo-first-order rate kinetics. A double-logarithmic plot of the apparent reaction rate constant against FITC concentration yielded a straight line with a slope of 0.89, suggesting that the alteration of a single lysine residue on the enzyme is sufficient to inhibit vacuolar H(+)-PPase. Changes in K(m) but not V(max) values of vacuolar H(+)-PPase as inhibited by FITC were obtained, indicating that the labeling caused a modification in affinity of the enzyme to its substrate. FITC inhibition of vacuolar H(+)-PPase could be protected by its physiological substrate, Mg(2+)-PP(i). These results indicate that FITC might specifically compete with the substrate at the active site and the FITC-labeled lysine residue locates probably in or near the catalytic domain of the enzyme. The enhancement of fluorescence intensity and the blue shift of the emission maximum of FITC after modification of vacuolar H(+)-PPase suggest that the FITC-labeled lysine residue is located in a relatively hydrophobic region.     

A lysine substitute for K+. A460K mutation eliminates K+ dependence in H+-pyrophosphatase of Carboxydothermus hydrogenoformans

The H(+) proton-translocating inorganic pyrophosphatase (H(+)-PPase) family is composed of two phylogenetically distinct types of enzymes: K(+)-dependent and K(+)-independent. However, to date, the sequence criteria governing this dichotomy have remained unknown. In this study, we describe the heterologous expression and functional characterization of H(+)-PPase from the thermophilic bacterium Carboxydothermus hydrogenoformans. Both PP(i)-hydrolyzing and PP(i)-energized H(+) translocation activities of the recombinant enzyme in Escherichia coli inner membrane vesicles are strictly K(+)-dependent. Here we deduce the K(+) requirement of all available H(+)-PPase sequences based on the K(+) dependence of C. hydrogenoformans H(+)-PPase in conjunction with phylogenetic analyses. Our data reveal that K(+)-independent H(+)-PPases possess conserved Lys and Thr that are absent in K(+)-dependent H(+)-PPases. We further demonstrate that a A460K substitution in C. hydrogenoformans H(+)-PPase is sufficient to confer K(+) independence to both PP(i) hydrolysis and PP(i)-energized H(+) translocation. In contrast, a A463T mutation does not affect the K(+) dependence of H(+)-PPase.     

H+-pyrophosphatase of Rhodospirillum rubrum. High yield expression in Escherichia coli and identification of the Cys residues responsible for inactivation my mersalyl

H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.     

硅改善盐胁迫下库拉索芦荟生长和离子吸收与分布

Si2．0mmol／L处理明显缓解NaCl 100、200mmol／L胁迫120d对库拉索芦荟（Aloevera）生长的抑制作用。Si可显著降低NaCl胁迫下芦荟植株中的Na^＋和Cl^-含量,提高K^＋含量,从而显著降低K^＋／Na^＋,促进根对K^＋的选择性吸收（ASK,Na）和K^＋向地上部的选择性运输（TSK,Na）,以维持植株体内的离子稳态。根系和叶片横切面的X-射线能谱微区分析结果进一步证实了这一结果。Si改善盐胁迫下芦荟对K^＋的选择性吸收和运输的机制之一是通过显著提高盐胁迫下芦荟根细胞质膜H^＋ATPase、液泡膜H^＋-ATPase和液泡膜H^＋-PPase的活性。     

Expression of functional Streptomyces coelicolor H+-pyrophosphatase and characterization of its molecular properties

H(+)-translocating pyrophosphatases (H(+)-PPases) are proton pumps that are found in many organisms, including plants, bacteria and protozoa. Streptomyces coelicolor is a soil bacterium that produces several useful antibiotics. Here we investigated the properties of the H(+)-PPase of S. coelicolor by expressing a synthetic DNA encoding the amino-acid sequence of the H(+)-PPase in Escherichia coli. The H(+)-PPase from E. coli membranes was active at a relatively high pH, stable up to 50 degrees C, and sensitive to N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide and acylspermidine. Enzyme activity increased by 60% in the presence of 120 mM K(+), which was less than the stimulation observed with plant vacuolar H(+)-PPases (type I). Substitutions of Lys-507 in the Gly-Gln-x-x-(Ala/Lys)-Ala motif, which is thought to determine the K(+) requirement of H(+)-PPases, did not alter its K(+) dependence, suggesting that other residues control this feature of the S. coelicolor enzyme. The H(+)-PPase was detected during early growth and was present mainly on the plasma membrane and to a lesser extent on intracellular membranous structures.     

Vacuolar H(+)-pyrophosphatase

The H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) is a unique, electrogenic proton pump distributed among most land plants, but only some alga, protozoa, bacteria, and archaebacteria. This enzyme is a fine model for research on the coupling mechanism between the pyrophosphate hydrolysis and the active proton transport, since the enzyme consists of a single polypeptide with a calculated molecular mass of 71-80 kDa and its substrate is also simple. Cloning of the H(+)-PPase genes from several organisms has revealed the conserved regions that may be the catalytic site and/or participate in the enzymatic function. The primary sequences are reviewed with reference to biochemical properties of the enzyme, such as the requirement of Mg(2)(+) and K(+). In plant cells, H(+)-PPase coexists with H(+)-ATPase in a single vacuolar membrane. The physiological significance and the regulation of the gene expression of H(+)-PPase are also reviewed.     

Oligomerization of H(+)-pyrophosphatase and its structural and functional consequences

The H(+)-pyrophosphatase (H(+)-PPase) consists of a single polypeptide, containing 16 or 17 transmembrane domains. To determine the higher order oligomeric state of Streptomyces coelicolor H(+)-PPase, we constructed a series of cysteine substitution mutants and expressed them in Escherichia coli. Firstly, we analyzed the formation of disulfide bonds, promoted by copper, in mutants with single cysteine substitutions. 28 of 39 mutants formed disulfide bonds, including S545C, a substitution at the periplasmic side. The formation of intermolecular disulfide bonds suppressed the enzyme activity of several, where the substituted residues were located in the cytosol. Creating disulfide links in the cytosol may interfere with the enzyme's catalytic function. Secondly, we prepared double mutants by introducing second cysteine substitutions into the S545C mutant. These double-cysteine mutants produced cross-linked complexes, estimated to be at least tetramers and possibly hexamers. Thirdly, we co-expressed epitope-tagged, wild type, and inactive mutant H(+)-PPases in E. coli and confirmed the formation of oligomers by co-purifying one subunit using the epitope tag used to label the other. The enzyme activity of these oligomers was markedly suppressed. We propose that H(+)-PPase is present as an oligomer made up of at least two or three sets of dimers.     

A plant-like vacuolar H(+)-pyrophosphatase in Plasmodium falciparum.

Inorganic pyrophosphate promoted the acidification of a subcellular compartment in cell homogenates of Plasmodium falciparum trophozoites. The proton gradient driven by pyrophosphate was collapsed by addition of NH(4)Cl or the K(+)/H(+) exchanger nigericin and eliminated by the pyrophosphate analog aminomethylenediphosphonate. Pyrophosphatase activity was dependent upon K(+), and partially inhibited by Na(+). The presence of a plant-like vacuolar H(+)-translocating pyrophosphatase (V-H(+)-PPase) was confirmed using antibodies raised against conserved peptide sequences of the enzyme, which cross reacted with a protein band of 76.5 kDa. Immunofluorescence microscopy using these antibodies showed a general fluorescence over the whole parasites and intracellular bright spots suggesting a vesicular and plasma membrane localization. Together, these results indicate the presence in P. falciparum of a V-H(+)-PPase of similar characteristics to those of the enzyme from plants.     

Overexpression of an H+-PPase gene from Thellungiella halophila in cotton enhances salt tolerance and improves growth and photosynthetic performance

Salinity is one of the major environmental factors limiting plant growth and productivity. An H(+)-PPase gene, TsVP from Thellungiella halophila, was transferred into cotton (Gossypium hirsutum) in sense and antisense orientations under control of the cauliflower mosaic virus (CaMV) 35S promoter. Southern and Northern blotting analysis showed that the sense or antisense TsVP were integrated into the cotton genome and expressed. Transgenic plants overexpressing the vacuolar H(+)-PPase were much more resistant to 150 and 250 mM NaCl than the isogenic wild-type plants. In contrast, the plants from the antisense line (L-2), with lower H(+)-PPase activity, were more sensitive to salinity than the wild-type plants. Overexpressing TsVP in cotton improved shoot and root growth and photosynthetic performance. These transgenic plants accumulated more Na(+), K(+), Ca(2+), Cl(-) and soluble sugars in their root and leaf tissues under salinity conditions compared with the wild-type plants. The lower membrane ion leakage and malondialdehyde (MDA) level in these transgenic plants suggest that overexpression of H(+)-PPase causes the accumulation of Na(+) and Cl(-) in vacuoles instead of in the cytoplasm, thus reducing their toxic effects. On the other hand, the increased accumulation of ions and sugars decreases the solute potential in cells, and facilitates water uptake under salinity, which is an important mechanism for the increased salt tolerance in TsVP-overexpressing cotton.     

Properties of mutated Rhodospirillum rubrum H+-pyrophosphatase expressed in Escherichia coli

The membrane-bound proton pumping inorganic pyrophosphate synthase/pyrophosphatase (H(+)-PPi synthase/H(+)-PPase) from the photosynthetic bacterium Rhodospirillum rubrum was functionally expressed in Escherichia coli C43(DE3) cells. Based on a new topology model of the enzyme, charged residues predicted to be located near or within the membrane were selected for site-directed mutagenesis. Several of these mutations resulted in an almost complete inactivation of the enzyme. Four mutated residues appear to show a selective impairment of proton translocation and are thus likely to be involved in coupling pyrophosphate hydrolysis with electrogenic proton pumping. Two of these mutations, R176K and E584D, caused increased tolerance to salt. In addition, the former mutation caused an increased K(m) of one order of magnitude for the hydrolysis reaction. These results and their possible implications for the enzyme function are discussed.     

Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity

Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima(Tm-PPase), a homologue of H(+)-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H(+)-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na(+) but displayed the highest activity in the presence of millimolar levels of both Na(+) and K(+). Detergent-solubilized recombinant Tm-PPase was thermostable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady-state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na(+) binding sites and one K(+) binding site are involved in enzyme activation. The affinity of the site that binds Na(+) first is increased with increasing K(+) concentration. In contrast, only one Na(+) binding site (K(+)-dependent) and one K(+) binding site were involved in activation of the Asp(703) --> Asn variant. Thus, Asp(703) may form part of the K(+)-independent Na(+) binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PP(i)-synthesizing activity, which also required Na(+) but was inhibited by K(+). These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na(+)-dependent H(+)-PPase homologues and may be an analogue of Na(+),K(+)-ATPase.     

Thermoinactivation analysis of vacuolar H(+)-pyrophosphatase

Vacuolar H(+)-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) catalyzes both the hydrolysis of PP(i) and the electrogenic translocation of proton from the cytosol to the lumen of the vacuole. Vacuolar H(+)-PPase, purified from etiolated hypocotyls of mung bean (Vigna radiata L.), is a homodimer with a molecular mass of 145 kDa. To investigate the relationship between structure and function of this H(+)-translocating enzyme, thermoinactivation analysis was employed. Thermoinactivation studies suggested that vacuolar H(+)-PPase consists of two distinct states upon heat treatment and exhibited different transition temperatures in the presence and absence of ligands (substrate and inhibitors). Substrate protection of H(+)-PPase stabilizes enzyme structure by increasing activation energy from 54.9 to 70.2 kJ/mol. We believe that the conformation of this enzyme was altered in the presence of substrate to protect against the thermoinactivation. In contrast, the modification of H(+)-PPase by inhibitor (fluorescein 5'-isothiocyanate; FITC) augmented the inactivation by heat treatment. The native, substrate-bound, and FITC-labeled vacuolar H(+)-PPases possess probably distinct conformation and show different modes of susceptibility to thermoinactivation. Our results also indicate that the structure of one subunit of this homodimer exerts long distance effect on the other, suggesting a specific subunit-subunit interaction in vacuolar H(+)-PPase. A working model was proposed to interpret the relationship of the structure and function of vacuolar H(+)-PPase.     

Chimeric domain analysis of the compatibility between H(+), K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits for the functional expression of gastric H(+),K(+)-ATPase.

Gastric H(+),K(+)-ATPase consists of alpha-subunit with 10 transmembrane domains and beta-subunit with a single transmembrane domain. We constructed cDNAs encoding chimeric beta-subunits between the gastric H(+),K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits and co-transfected them with the H(+),K(+)-ATPase alpha-subunit cDNA in HEK-293 cells. A chimeric beta-subunit that consists of the cytoplasmic plus transmembrane domains of Na(+),K(+)-ATPase beta-subunit and the ectodomain of H(+),K(+)-ATPase beta-subunit assembled with the H(+),K(+)-ATPase alpha-subunit and expressed the K(+)-ATPase activity. Therefore, the whole cytoplasmic and transmembrane domains of H(+),K(+)-ATPase beta-subunit were replaced by those of Na(+),K(+)-ATPase beta-subunit without losing the enzyme activity. However, most parts of the ectodomain of H(+),K(+)-ATPase beta-subunit were not replaced by the corresponding domains of Na(+), K(+)-ATPase beta-subunit. Interestingly, the extracellular segment between Cys(152) and Cys(178), which contains the second disulfide bond, was exchangeable between H(+),K(+)-ATPase and Na(+), K(+)-ATPase, preserving the K(+)-ATPase activity intact. Furthermore, the K(+)-ATPase activity was preserved when the N-terminal first 4 amino acids ((67)DPYT(70)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the corresponding amino acids ((63)SDFE(66)) of Na(+),K(+)-ATPase beta-subunit. The ATPase activity was abolished, however, when 4 amino acids ((76)QLKS(79)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the counterpart ((72)RVAP(75)) of Na(+),K(+)-ATPase beta-subunit, indicating that this region is the most N-terminal one that discriminates the H(+),K(+)-ATPase beta-subunit from that of Na(+), K(+)-ATPase.     

Role of hyperpolarization-activated currents for the intrinsic dynamics of isolated retinal neurons

The intrinsic dynamics of bipolar cells and rod photoreceptors isolated from tiger salamanders were studied by a patch-clamp technique combined with estimation of effective impulse responses across a range of mean membrane voltages. An increase in external K(+) reduces the gain and speeds the response in bipolar cells near and below resting potential. High external K(+) enhances the inward rectification of membrane potential, an effect mediated by a fast, hyperpolarization-activated, inwardly rectifying potassium current (K(IR)). External Cs(+) suppresses the inward-rectifying effect of external K(+). The reversal potential of the current, estimated by a novel method from a family of impulse responses below resting potential, indicates a channel that is permeable predominantly to K(+). Its permeability to Na(+), estimated from Goldman-Hodgkin-Katz voltage equation, was negligible. Whereas the activation of the delayed-rectifier K(+) current causes bandpass behavior (i.e., undershoots in the impulse responses) in bipolar cells, activation of the K(IR) current does not. In contrast, a slow hyperpolarization-activated current (I(h)) in rod photoreceptors leads to pronounced, slow undershoots near resting potential. Differences in the kinetics and ion selectivity of hyperpolarization-activated currents in bipolar cells (K(IR)) and in rod photoreceptors (I(h)) confer different dynamical behavior onto the two types of neurons.     

Roles of histidine residues in plant vacuolar H(+)-pyrophosphatase

Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis. Alignment analysis on amino acid sequence demonstrates that vacuolar H(+)-PPase of mung bean contains six highly conserved histidine residues. Previous evidence indicated possible involvement of histidine residue(s) in enzymatic activity and H(+)-translocation of vacuolar H(+)-PPase as determined by using histidine specific modifier, diethylpyrocarbonate [J. Protein Chem. 21 (2002) 51]. In this study, we further attempted to identify the roles of histidine residues in mung bean vacuolar H(+)-PPase by site-directed mutagenesis. A line of mutants with histidine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Among the mutants scrutinized, only the mutation of H716 significantly decreased the enzymatic activity, the proton transport, and the coupling ratio of vacuolar H(+)-PPase. The enzymatic activity of H716A is relatively resistant to inhibition by diethylpyrocarbonate as compared to wild-type and other mutants, indicating that H716 is probably the target residue for the attack by this modifier. The mutation at H716 of V-PPase shifted the optimum pH value but not the T(1/2) (pretreatment temperature at which half enzymatic activity is observed) for PP(i) hydrolytic activity. Mutation of histidine residues obviously induced conformational changes of vacuolar H(+)-PPase as determined by immunoblotting analysis after limited trypsin digestion. Furthermore, mutation of these histidine residues modified the inhibitory effects of F(-) and Na(+), but not that of Ca(2+). Single substitution of H704, H716 and H758 by alanine partially released the effect of K(+) stimulation, indicating possible location of K(+) binding in the vicinity of domains surrounding these residues.     

Alkali cation binding and permeation in the rat organic cation transporter rOCT2

Organic cation transporters of the OCT family mediate downhill transport of organic cations, compatible with carrier, pore, or gate-lumen-gate mechanisms. We studied rat OCT2 expressed in Xenopus oocytes by the two-electrode voltage-clamp technique, including membrane capacitance (C(m)) monitoring. Choline, a transported cationic substrate, elicited the expected inward currents but also elicited decreases of C(m). Similar C(m) decreases were caused by the non-transported inhibitors tetrabutylammonium (a cation) and corticosterone (uncharged). Effects on C(m) were voltage-dependent, with a maximum at -140 mV. These findings suggest that the empty rOCT2 protein can undergo an electrogenic conformation change, with one conformation highly favored at physiological voltage. Moreover, alkali cations elicited considerable inward currents and inhibited uptake of [(14)C]tetraethylammonium with a sequence Cs(+) > Rb(+) > K(+) > Na(+) approximately Li(+). Cs(+) affected current and capacitance with similar affinity (K(0.5) approximately 50 mm). Tetraethylammonium inhibited Cs(+) currents in a concentration-dependent manner. Conversely, Cs(+) inhibited tetraethylammonium uptake by a competitive mechanism. Activation energy of the currents estimated from measurements between 12 degrees C and 32 degrees C was approximately 81 kJ/mol for Cs(+) and 39 kJ/mol for tetramethylammonium, compatible with permeation of Cs(+) through rOCT2 along the same path as organic substrates and by a mechanism different from simple electrodiffusion. Rationalization of Cs(+) selectivity in terms of a pore pointed to a pore diameter of approximately 4 A. Intriguingly, that value matches the known selectivity of rOCT2 for organic compounds. Our data show that selective permeability of rOCT2 is not determined by ligand affinity but might rather be understood in terms of the ion channel concept of a distinct "selectivity filter."     

Vacuolar H(+)-translocating pyrophosphatases: a new category of ion translocase.

The membrane surrounding the central vacuole of plant cells contains an H(+)-translocating ATPase (H(+)-ATPase) and an H(+)-translocating inorganic pyrophosphatase (H(+)-PPase). Both enzymes are abundant and ubiquitous in plants but the H(+)-PPase is unusual in its exclusive use of inorganic pyrophosphate (PPi) as an energy source. The lack of sequence identity between the vacuolar H(+)-PPase and any other characterized ion pump implies a different evolutionary origin for this translocase. The existence of the vacuolar H(+)-PPase, in conjunction with increasing recognition of PPi as a key metabolite in plant systems, necessitates reconsideration of ATP as the primary energy source for membrane transport in plant cells.     

H2O2 activates redox- and 4-aminopyridine-sensitive Kv channels in coronary vascular smooth muscle

Previously, we demonstrated that coronary vasodilation in response to hydrogen peroxide (H(2)O(2)) is attenuated by 4-aminopyridine (4-AP), an inhibitor of voltage-gated K(+) (K(V)) channels. Using whole cell patch-clamp techniques, we tested the hypothesis that H(2)O(2) increases K(+) current in coronary artery smooth muscle cells. H(2)O(2) increased K(+) current in a concentration-dependent manner (increases of 14 +/- 3 and 43 +/- 4% at 0 mV with 1 and 10 mM H(2)O(2), respectively). H(2)O(2) increased a conductance that was half-activated at -18 +/- 1 mV and half-inactivated at -36 +/- 2 mV. H(2)O(2) increased current amplitude; however, the voltages of half activation and inactivation were not altered. Dithiothreitol, a thiol reductant, reversed the effect of H(2)O(2) on K(+) current and significantly shifted the voltage of half-activation to -10 +/- 1 mV. N-ethylmaleimide, a thiol-alkylating agent, blocked the effect of H(2)O(2) to increase K(+) current. Neither tetraethylammonium (1 mM) nor iberiotoxin (100 nM), antagonists of Ca(2+)-activated K(+) channels, blocked the effect of H(2)O(2) to increase K(+) current. In contrast, 3 mM 4-AP completely blocked the effect of H(2)O(2) to increase K(+) current. These findings lead us to conclude that H(2)O(2) increases the activity of 4-AP-sensitive K(V) channels. Furthermore, our data support the idea that 4-AP-sensitive K(V) channels are redox sensitive and contribute to H(2)O(2)-induced coronary vasodilation.