NADPH oxidase does not account fully for O2-sensing in model airway chemoreceptor cells

A key feature of O2 sensing by chemoreceptor tissues is the hypoxic inhibition of K+ channels. However, mechanisms coupling a fall of pO2 to channel closure differ between tissues: O2 regulation of K+ channels in chemoreceptive neuroepithelial bodies and their immortal counterparts, H146 cells, involves altered reactive oxygen species generation by NADPH oxidase. In contrast, this enzyme complex is not involved in O2 sensing by the carotid body and pulmonary vasculature. Here, we provide pharmacological evidence to support a role for NADPH oxidase in hypoxic inhibition of K+ currents in H146 cells. Two structurally unrelated NADPH oxidase inhibitors, diphenylene iodonium and phenylarsine oxide, suppressed hypoxic inhibition of K+ currents recorded using the patch-clamp technique. Most importantly, however, neither inhibitor fully blocked this response. Our findings provide the first evidence that multiple mechanisms may coexist within a specific cell type to account for hypoxic suppression of K+ channel activity.     

Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Large-conductance Ca²⁺-activated K⁺ (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK^–/–) mice, we stimulated NADPH oxidase activity with 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O₂^– and H₂O₂ production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn²⁺, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca²⁺ and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca²⁺ by N-formyl-L-methionyl-L-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK^–/– and BK^+/+ mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity. killing assay; reactive oxygen species; BK-deficient mice; mice infection     

Interactions between electron and proton currents in excised patches from human eosinophils

The NADPH-oxidase is a plasma membrane enzyme complex that enables phagocytes to generate superoxide in order to kill invading pathogens, a critical step in the host defense against infections. The oxidase transfers electrons from cytosolic NADPH to extracellular oxygen, a process that requires concomitant H+ extrusion through depolarization-activated H+ channels. Whether H+ fluxes are mediated by the oxidase itself is controversial, but there is a general agreement that the oxidase and H+ channel are intimately connected. Oxidase activation evokes profound changes in whole-cell H+ current (IH), causing an approximately -40-mV shift in the activation threshold that leads to the appearance of inward IH. To further explore the relationship between the oxidase and proton channel, we performed voltage-clamp experiments on inside-out patches from both resting and phorbol-12-myristate-13-acetate (PMA)-activated human eosinophils. Proton currents from resting cells displayed slow voltage-dependent activation, long-term stability, and were blocked by micromolar internal [Zn2+]. IH from PMA-treated cells activated faster and at lower voltages, enabling sustained H+ influx, but ran down within minutes, regaining the current properties of nonactivated cells. Bath application of NADPH to patches excised from PMA-treated cells evoked electron currents (Ie), which also ran down within minutes and were blocked by diphenylene iodonium (DPI). Run-down of both IH and Ie was delayed, and sometimes prevented, by cytosolic ATP and GTP-gamma-S. A good correlation was observed between the amplitude of Ie and both inward and outward IH when a stable driving force for e- was imposed. Combined application of NADPH and DPI reduced the inward IH amplitude, even in the absence of concomitant oxidase activity. The strict correlation between Ie and IH amplitudes and the sensitivity of IH to oxidase-specific agents suggest that the proton channel is either part of the oxidase complex or linked by a membrane-limited mediator.     

The gp91phox component of NADPH oxidase is not the voltage-gated proton channel in phagocytes, but it helps

During the "respiratory burst," the NADPH oxidase complex of phagocytes produces reactive oxygen species that kill bacteria and other invaders (Babior, B. M. (1999) Blood 93, 1464-1476). Electron efflux through NADPH oxidase is electrogenic (Henderson, L. M., Chappell, J. B., and Jones, O. T. G. (1987) Biochem. J. 246, 325-329) and is compensated by H(+) efflux through proton channels that reportedly are contained within the gp91(phox) subunit of NADPH oxidase. To test whether gp91(phox) functions as a proton channel, we studied H(+) currents in granulocytes from X-linked chronic granulomatous disease patients lacking gp91(phox) (X-CGD), the human myelocytic PLB-985 cell line, PLB-985 cells in which gp91(phox) was knocked out by gene targeting (PLB(KO)), and PLB-985 knockout cells re-transfected with gp91(phox) (PLB(91)). H(+) currents in unstimulated PLB(KO) cells had amplitude and gating kinetics similar to PLB(91) cells. Furthermore, stimulation with the phorbol ester phorbol 12-myristate 13-acetate increased H(+) currents to a similar extent in X-CGD, PLB(KO), and PLB(91) cells. Thus, gp91(phox) is not the proton channel in unstimulated phagocytes and does not directly mediate the increase of proton conductance during the respiratory burst. Changes in H(+) channel gating kinetics during NADPH oxidase activity are likely crucial to the activation of H(+) flux during the respiratory burst.     

Lipopolysaccharide-sensitive H+ current in dendritic cells

Dendritic cells (DCs) are the most potent antigen-presenting cells equipped to transport antigens from the periphery to lymphoid tissues and to present them to T cells. Ligation of Toll-like receptor 4 (TLR4), expressed on the DC surface, by lipopolysaccharides (LPS), elements of the Gram-negative bacteria outer wall, induces DC maturation. Initial steps of maturation include stimulation of antigen endocytosis and enhanced reactive oxygen species (ROS) production with eventual downregulation of endocytic capacity in fully matured DCs. ROS production depends on NADPH oxidase (NOX2), the activity of which requires continuous pH and charge compensation. The present study demonstrates, for the first time, the functional expression of voltage-gated proton (Hv1) channels in mouse bone marrow-derived DCs. In whole cell patch-clamp experiments, we recorded Zn(2+) (50 μM)-sensitive outwardly rectifying currents activated upon depolarization, which were highly selective for H(+), with the reversal potential shift of 38 mV per pH unit. The threshold voltage of activation (V(threshold)) was dependent on the pH gradient and was close to the empirically predicted V(threshold) for the Hv1 currents. LPS (1 μg/ml) had bimodal effects on Hv1 channels: acute LPS treatment increased Hv1 channel activity, whereas 24 h of LPS incubation significantly inhibited Hv1 currents and decreased ROS production. Activation of H(+) currents by acute application of LPS was abolished by PKC inhibitor GFX (10 nM). According to electron current measurements, acute LPS application was associated with increased NOX2 activity.     

Heme histidine ligands within gp91(phox) modulate proton conduction by the phagocyte NADPH oxidase

The membrane subunit of the phagocyte NADPH oxidase, gp91(phox), possesses a H(+) channel motif formed by membrane-spanning histidines postulated to coordinate the two heme groups forming the redox center of the flavocytochrome. To study the role of heme-binding histidines on proton conduction, we stably expressed the gp91(phox) cytochrome in human embryonic kidney 293 cells and measured proton currents with the patch clamp technique. Similar to its shorter homologue, NADPH oxidase homologue 1, which is predicted not to bind heme, gp91(phox) generated voltage-activated, pH-dependent, H(+)-selective currents that were reversibly blocked by Zn(2+). The gp91(phox) currents, however, activated faster, deactivated more slowly, and were markedly affected by the inhibition of heme synthesis. Upon heme removal, the currents had larger amplitude, activated faster and at lower voltages, and became sensitive to the histidine reagent diethylpyrocarbonate. Mutation of the His-115 residue to leucine abolished both the gp91(phox) characteristic 558-nm absorbance peak and voltage-activated currents, indicating that His-115 is involved in both heme ligation and proton conduction. These results indicate that the gp91(phox) proton channel is activated upon release of heme from its His-115 ligand. During activation of the oxidase complex, changes in heme coordination within the cytochrome might increase the mobility of histidine ligands, thereby coupling electron and proton transport.     

Absence of proton channels in COS-7 cells expressing functional NADPH oxidase components

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O(2)(-)) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H(+) efflux was thought to be contained within the gp91(phox) subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063-36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COS(phox)). The 7D5 antibody, which detects an extracellular epitope of the gp91(phox) protein, labeled 96-98% of COS(phox) cells. NADPH oxidase was functional because COS(phox) (but not COS(WT)) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COS(WT)) or COS(phox) cells studied at pH(o) 7.0 and pH(i) 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells. Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.     

Interactions between NADPH oxidase and voltage-gated proton channels: why electron transport depends on proton transport

Leukocytes kill microbes by producing reactive oxygen species, using a multi-component enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Electrons pass from intracellular NADPH through a redox chain within the enzyme, to reduce extracellular O2 to O2-. Electron flux is electrogenic, and rapidly depolarizes the membrane potential. Excessive depolarization can turn off electron transport by self-inhibition, but this is prevented by proton flux that balances the electron flux. Although the membrane potential depolarizes by approximately 100 mV during the respiratory burst (NADPH oxidase activity), NADPH oxidase activity is independent of voltage in this range, which permits optimal function and prevents self-inhibition.     

The antibacterial activity of human neutrophils and eosinophils requires proton channels but not BK channels

Electrophysiological events are of central importance during the phagocyte respiratory burst, because NADPH oxidase is electrogenic and voltage sensitive. We investigated the recent suggestion that large-conductance, calcium-activated K(+) (BK) channels, rather than proton channels, play an essential role in innate immunity (Ahluwalia, J., A. Tinker, L.H. Clapp, M.R. Duchen, A.Y. Abramov, S. Page, M. Nobles, and A.W. Segal. 2004. Nature. 427:853-858). In PMA-stimulated human neutrophils or eosinophils, we did not detect BK currents, and neither of the BK channel inhibitors iberiotoxin or paxilline nor DPI inhibited any component of outward current. BK inhibitors did not inhibit the killing of bacteria, nor did they affect NADPH oxidase-dependent degradation of bacterial phospholipids by extracellular gIIA-PLA(2) or the production of superoxide anion (O(2*)(-)). Moreover, an antibody against the BK channel did not detect immunoreactive protein in human neutrophils. A required role for voltage-gated proton channels is demonstrated by Zn(2+) inhibition of NADPH oxidase activity assessed by H(2)O(2) production, thus validating previous studies showing that Zn(2+) inhibited O(2*)(-) production when assessed by cytochrome c reduction. In conclusion, BK channels were not detected in human neutrophils or eosinophils, and BK inhibitors did not impair antimicrobial activity. In contrast, we present additional evidence that voltage-gated proton channels serve the essential role of charge compensation during the respiratory burst.     

Voltage-gated proton channel is expressed on phagosomes

Voltage-gated proton channel has been suggested to help NADPH oxidase activity during respiratory burst of phagocytes through its activities of compensating charge imbalance and regulation of pH. In phagocytes, robust production of reactive oxygen species occurs in closed membrane compartments, which are called phagosomes. However, direct evidence for the presence of voltage-gated proton channels in phagosome has been lacking. In this study, the expression of voltage-gated proton channels was studied by Western blot with the antibody specific to the voltage-sensor domain protein, VSOP/Hv1, that has recently been identified as the molecular correlate for the voltage-gated proton channel. Phagosomal membranes of neutrophils contain VSOP/Hv1 in accordance with subunits of NADPH oxidases, gp91, p22, p47 and p67. Superoxide anion production upon PMA activation was significantly reduced in neutrophils from VSOP/Hv1 knockout mice. These are consistent with the idea that voltage-gated proton channels help NADPH oxidase in phagocytes to produce reactive oxygen species.     

Currents through Hv1 channels deplete protons in their vicinity

Proton channels have evolved to provide a pH regulatory mechanism, affording the extrusion of protons from the cytoplasm at all membrane potentials. Previous evidence has suggested that channel-mediated acid extrusion could significantly change the local concentration of protons in the vicinity of the channel. In this work, we directly measure the proton depletion caused by activation of Hv1 proton channels using patch-clamp fluorometry recordings from channels labeled with the Venus fluorescent protein at intracellular domains. The fluorescence of the Venus protein is very sensitive to pH, thus behaving as a genetically encoded sensor of local pH. Eliciting outward proton currents increases the fluorescence intensity of Venus. This dequenching is related to the magnitude of the current and not to channel gating and is dependent on the pH gradient. Our results provide direct evidence of local proton depletion caused by flux through the proton-selective channel.     

Regulation of human eosinophil NADPH oxidase activity: a central role for PKCdelta.

Eosinophils play a primary role in the pathophysiology of asthma. In the lung, the activation state of the infiltrating eosinophils determines the extent of tissue damage. Interleukin-5 (IL-5) and leukotriene B4 (LTB4) are important signaling molecules involved in eosinophil recruitment and activation. However, the physiological processes that regulate these activation events are largely unknown. In this study we have examined the mechanisms of human eosinophil NADPH oxidase regulation by IL-5, LTB4, and phorbol ester (PMA). These stimuli activate a Zn2+-sensitive plasma membrane proton channel, and treatment of eosinophils with Zn2+ blocks superoxide production. We have demonstrated that eosinophil intracellular pH is not altered by IL-5 activation of NADPH oxidase. Additionally, PKCdelta inhibitors block PMA, IL-5 and LTB4 mediated superoxide formation. Interestingly, the PKCdelta-selective inhibitor, rottlerin, does not block proton channel activation by PMA indicating that the oxidase and the proton conductance are regulated at distinct phosphorylation sites. IL-5 and LTB4, but not PMA, stimulated superoxide production is also blocked by inhibitors of PI 3-kinase indicating that activation of this enzyme is an upstream event common to both receptor signaling pathways. Our results indicate that the G-protein-coupled LTB4 receptor and the IL-5 cytokine receptor converge on a common signaling pathway involving PI 3-kinase and PKCdelta to regulate NADPH oxidase activity in human eosinophils.     

Characterisation of electron currents generated by the human neutrophil NADPH oxidase

Electron transport by the human neutrophil NADPH oxidase is an important microbicidal weapon for phagocytes. The electron current (I_e) generated by the neutrophil NADPH oxidase is poorly characterised due to the lack of appropriate electrophysiological data. In this study, I fully characterise the neutrophil generated I_e when the NADPH oxidase is activated by NADPH and GTPγS. The neutrophil I_e was markedly voltage-dependent in the entire voltage range in comparison to those electron currents measured after chloride was removed from the external bath solution. The difference in I_e measured in chloride free conditions was not due to a change in the activation kinetics of voltage-gated proton channels. The I_e depolarises the neutrophil plasma membrane at a rate of 2.3 V s⁻¹ and this depolarisation was opposed when voltage-gated proton channels are activated. 3 mM ZnCl₂ depolarised the membrane potential to +97.8 ± 2.5 mV (n = 4), and this depolarisation was abolished after NADPH oxidase inhibition.     

Replacing Asn207 by aspartate at the neck of the D channel in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides results in decoupling the proton pump

Cytochrome oxidase catalyzes the reduction of O2 to water and conserves the considerable free energy available from this reaction in the form of a proton motive force. For each electron, one proton is electrogenically pumped across the membrane. Of particular interest is the mechanism by which the proton pump operates. Previous studies of the oxidase from Rhodobacter sphaeroides have shown that all of the pumped protons enter the enzyme through the D channel and that a point mutant, N139D, in the D channel completely eliminates proton pumping without reducing oxidase activity. N139 is one of three asparagines near the entrance of the D channel, where there is a narrowing or neck, through which a single file of water molecules pass. In the current work, it is shown that replacement of a second asparagine in this region by an asparate, N207D, also decouples the proton pump without altering the oxidase activity of the enzyme. Previous studies demonstrated that the N139D mutant results in an increase in the apparent pKa of E286, a functionally critical residue that is located 20 A away from N139 at the opposite end of the D channel. In the current work, it is shown that the N207 mutation also increases the apparent pKa of E286. This finding reinforces the proposal that the elimination of proton pumping is the result of an increase of the apparent proton affinity of E286, which, in turn, prevents the timely proton transfer to a proton accepter group within the exit channel of the proton pump.     

Bupivacaine inhibits large conductance, voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K(+) channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca(2+) -activated K(+) channels (BK(Ca)). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K(+) currents carried by BK(Ca) channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC(50) 324 μM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 μM) can also block whole-cell K(+) currents (~45% blockage) in which, under our working conditions, BK(Ca) is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK(Ca) channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.     

Experimental aspects of isolation of myocytes from adult guinea-pig hearts

Ionic channels can now be effectively studied on enzymatically isolated cardiac myocytes by means of the patch clamp technique. Three procedures reported to give consistently high yields of Ca-tolerant myocytes were tested for applicability to calcium channel studies under our laboratory conditions. None of them was found to be suitable for direct use. Therefore, a modified method for isolation of myocytes from adult guinea-pig hearts was developed. Calcium channel currents measured in Ca-tolerant myocytes isolated by this procedure have been presented and problems of myocytes isolation and of patch-clamp measurement discussed.     

Role of nucleotides and phosphoinositides in the stability of electron and proton currents associated with the phagocytic NADPH oxidase

The phagocytic NADPH oxidase (phox) moves electrons across cell membranes to kill microbes. The activity of this lethal enzyme is tightly regulated, but the mechanisms that control phox inactivation are poorly understood for lack of appropriate assays. The phox generates measurable electron currents, I(e), that are associated with inward proton currents, I(H). To study the inactivation of the phox and of its associated proton channel, we determined which soluble factors can stabilize I(e) (induced by the addition of NADPH) and I(H) (initiated by small depolarizing voltage steps) in inside-out patches from PMA-activated human eosinophils. I(e) decayed rapidly in the absence of nucleotides (tau approximately 6 min) and was maximally stabilized by the combined addition of 5 mM ATP and 50 microM of the non-hydrolysable GTP analogue GTP[S] (guanosine 5'-[gamma-thio]triphosphate) (tau approximately 57 min), but not by either ATP or GTP[S] alone. I(H) also decayed rapidly and was stabilized by the ATP/GTP[S] mixture, but maximal stabilization of I(H) required further addition of 25 muM PI(3,4)P2 (phosphoinositide 3,4-bisphosphate) to the cytosolic side of the patch. PI(3,4)P2 had no effect on I(e) and its stabilizing effect on I(H) could not be mimicked by other phosphoinositides. Reducing the ATP concentration below millimolar levels decreased I(H) stability, an effect that was not prevented by phosphatase inhibitors but by the non-hydrolysable ATP analogue ATP[S] (adenosine 5'-[gamma-thio]triphosphate). Our data indicate that the assembled phox complex is very stable in eosinophil membranes if both ATP and GTP[S] are present, but inactivates within minutes if one of the nucleotides is removed. Stabilization of the phox-associated proton channel in a highly voltage-sensitive conformation does not appear to involve phosphorylation but ATP binding, and requires not only ATP and GTP[S] but also PI(3,4)P2, a protein known to anchor the cytosolic phox subunit p47(phox) to the plasma membrane.     

Simultaneous presence of p47(phox) and flavocytochrome b-245 are required for the activation of NADPH oxidase by anionic amphiphiles. Evidence for an intermediate state of oxidase activation.

We have examined the kinetics of NADPH oxidase activation induced by arachidonic acid or SDS in a cell-free system using mixtures of recombinant Phox proteins and purified flavocytochrome b-245. Activation of oxidase activity required the simultaneous presence of p47(phox), flavocytochrome b-245, and the anionic amphiphile. The activation of electron transfer reactions was much more rapid when iodonitrotetrazolium violet was used as electron acceptor than when oxygen alone was the acceptor. We propose that this difference represents an intermediate activation state of NADPH oxidase in which electron flow can proceed from NADPH to enzyme flavin (and hence to iodonitrotetrazolium violet) but not from flavin to heme (or not between the hemes). A model for NADPH oxidase activation is presented that is consistent with these observations.     

Bupivacaine inhibits large conductance,voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K⁺ channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca²⁺-activated K⁺ channels (BK_Ca). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K⁺ currents carried by BK_Ca channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC₅₀ 324 µM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 µM) can also block whole-cell K⁺ currents (~45% blockage) in which, under our working conditions, BK_Ca is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK_Ca channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.     

Differential expression and regulation of K(+) channels in the maize coleoptile: molecular and biophysical analysis of cells isolated from cortex and vasculature

Recently, two K(+) channel genes, ZMK1 and ZMK2, were isolated from maize coleoptiles. They are expressed in the cortex and vasculature, respectively. Expression in Xenopus oocytes characterized ZMK1 as an inwardly rectifying K(+) channel activated by external acidification, while ZMK2 mediates voltage-independent and proton-inhibited K(+) currents. In search of the related gene products in planta, we applied the patch-clamp technique to protoplasts isolated from the cortex and vasculature of Zea mays coleoptiles and mesocotyls. In the cortex, a 6-8 pS K(+) channel gave rise to inwardly rectifying K(+) currents. Like ZMK1, this channel was activated by apoplastic acidification. In contrast, protoplasts from vascular tissue expressing the sucrose transporter ZmSUT1 were dominated by largely voltage-independent K(+) currents with a single-channel conductance of 22 pS. The pronounced sensitivity to the extracellular protons Ca(2+), Cs(+) and Ba(2+) is reminiscent of ZMK2 properties in oocytes. Thus, the dominant K(+) channels in cortex and vasculature most likely represent the gene products of ZMK1 and ZMK2. Our studies on the ZMK2-like channels represent the first in planta analysis of a K+ channel that shares properties with the AKT3 K(+) channel family. Keywords: K(+) channel, voltage-independent, proton block, maize coleoptile.