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La Trobe University Faculty of Science, Technology and Engineering Murray Darling Freshwater Research CentreMDFRC item.The ability to measure directly individual protoplast ion fluxes is a valuable addition to patch clamp and other techniques when using protoplasts to study membrane transporters. Before interpreting observations on protoplasts in terms of behaviour of intact cells and tissues, some methodological questions should be addressed. These include effects of space and time variations of transporter activities over the membrane, the osmotic dependence of specific ion transporters and the effect of the regenerating cell wall. In this study net H+ and Ca2+ fluxes were measured from individual corn (Zea mays L.) coleoptile protoplasts using a noninvasive microelectrode technique for ion flux measurements. For Ca2+, the flux distribution was almost symmetrical, ranging +/- 30 nmol.m(-2).s(-1) around zero. For H+ it was skewed towards efflux ranging from -100 to + 10 nmol.m(-2).s(-1). The distribution of H+ fluxes through the protoplast surface was a complex mosaic which changed with time, sometimes showing oscillations. These flux variations with time and position around the surface, apparently driven by endogenous mechanisms, may be relevant to protoplast pH homeostasis. When the new cell wall was partially regenerated on the next day, the correlation between H+ and Ca2+ fluxes increased. which is consistent with the weak-acid Donnan-Manning model of cell wall ion exchange.

Calcium can ameliorate Na+ toxicity in plants by decreasing Na+ influx through nonselective cation channels. Here, we show that elevated external [Ca2+] also inhibits Na+-induced K+ efflux through outwardly directed, K+-permeable channels. Noninvasive ion flux measuring and patch-clamp techniques were used to characterize K+ fluxes from Arabidopsis (Arabidopsis thaliana) root mature epidermis and leaf mesophyll under various Ca2+ to Na+ ratios. NaCl-induced K+ efflux was not related to the osmotic component of the salt stress, was inhibited by the K+ channel blocker TEA+, was not mediated by inwardly directed K+ channels (tested in the akt1 mutant), and resulted in a significant decrease in cytosolic K+ content. NaCl-induced K+ efflux was partially inhibited by 1 mm Ca2+ and fully prevented by 10 mm Ca2+. This ameliorative effect was at least partially attributed to a less dramatic NaCl-induced membrane depolarization under high Ca2+ conditions. Patch-clamp experiments (whole-cell mode) have demonstrated that two populations of Ca2+-sensitive K+ efflux channels exist in protoplasts isolated from the mature epidermis of Arabidopsis root and leaf mesophyll cells. The instantaneously activating K+ efflux channels showed weak voltage dependence and insensitivity to external and internal Na+. Another population of K+ efflux channels was slowly activating, steeply rectifying, and highly sensitive to Na+. K+ efflux channels in roots and leaves showed different Ca2+ and Na+ sensitivities, suggesting that these organs may employ different strategies to withstand salinity. Our results suggest an additional mechanism of Ca2+ action on salt toxicity in plants: the amelioration of K+ loss from the cell by regulating (both directly and indirectly) K+ efflux channels.

Hypoxia-induced changes in net H +, K + and O 2 fluxes across the plasma membrane (PM) of epidermal root cells were measured using the non-invasive microelectrode ion flux measurement (MIFE) system in elongation, meristem and mature root zones of two barley (Hordeum vulgare L.) varieties contrasting in their waterlogging (WL) tolerance. The ultimate goal of this study was to shed light on the mechanisms underlying effects of WL on plant nutrient acquisition and mechanisms of WL tolerance in barley. Our measurements revealed that functionally different barley root zones have rather different O 2 requirements, with the highest O 2 influx being in the elongation zone of the root at about 1 mm from the tip. Oxygen deprivation has qualitatively different effects on the activity of PM ion transporters in mature and elongation zones. In the mature zone, hypoxic treatment caused a very sharp decline in K + uptake in the WL sensitive variety Naso Nijo, but did not reduce K + influx in the WL tolerant TX9425 variety. In the elongation zone, onset of hypoxia enhanced K + uptake from roots of both cultivars. Pharmacological experiments suggested that hypoxia-induced K + flux responses are likely to be mediated by both K + -inward- (KIR) and non-selective cation channels (NSCC) in the elongation zone, while in the mature zone K + -outward- (KOR) channels are the key contributors. Overall, our results suggest that oxygen deprivation has an immediate and substantial effect on root ion flux patterns, and that this effect is different in WL-sensitive and WL-tolerant cultivars. To what extent this difference in ion flux response to hypoxia is a factor conferring WL tolerance in barley remains to be answered in future studies. © 2006 Blackwell Publishing Ltd.

Potassium transport was investigated in the root elongation zone of Arabidopsis seedlings during the first minutes of Al3+ exposure, using the non-invasive MIFE microelectrode technique. To prevent pH changes during Al3+ application, and to separate aluminium from acidic stress, plants were pre-treated with 5 mM homoPIPES before addition of AlCl3 (pH 4.2). The 30-min treatment with 50 or 500 μM AlCl3 led to a significant increase in K+ efflux in solutions containing 100 μM CaCl2. This efflux was suppressed by high concentrations of Ca2+ (10 mM) in the bathing solution. Our results suggest that elevated external Ca2+ activities can sustain K+ influx in the root elongation zone during Al3+ exposure either by maintaining [Ca2+]cyt or by affecting Al3+ uptake across the plasma membrane.

Both parts of the actin-myosin complex involved in cytoplasmic streaming could be regulated by mineral ions. The main goal of this study was to find a relationship between cyclosis and ion transport across the cell wall and plasma membrane. The transport of K(+) and Ca(2+) along pH bands in Chara branchlet internodal cells was characterized by using the MIFE system for non-invasive microelectrode measurement of ion fluxes. Branchlets formed acidic and alkaline bands with the pH ranging from 5 to 8. Different pH patterns were observed for different sides of the branchlets. Sides with cyclosis streaming acropetally generally showed greater variation in the profiles of pH and H(+) fluxes. Although a high correlation was not found between pH bands and Ca(2+) or K(+) fluxes, there was a positive correlation between Ca(2+) and K(+) fluxes themselves for both sides of the branchlets. Application of cytochalasin D, an inhibitor of cyclosis, had no immediate effect on pH and ion fluxes, however, the time of cyclosis cessation corresponded with a dramatic change in Ca(2+) and K(+) fluxes; pH profiles and H(+) fluxes were affected within 2 h. The evidence suggests that, in Chara branchlets, pH band formation and Gd(3+)-insensitive Ca(2+) transport systems are linked to the cyclosis machinery: (i) the pH band amplitude for the acropetally streaming side was larger than that for the basipetally streaming side; (ii) cessation of cytoplasmic streaming after cytochalasin D application resulted in changed pH banding profiles and H(+), Ca(2+) and K(+) fluxes; and (iii) the application of GdCl(3) or incubation in GdCl(3) solutions did not lead to the cessation of cytoplasmic streaming, although external Ca(2+) fluxes changed.

Net fluxes of H a and Ca 2a were measured in the mesophyll tissue of broad bean (Vicia faba L.) leaves and in protoplasts derived from these cells. NaCl at 90 mM enhanced H a extrusion in both protoplasts and tissue, but in diAerent ways. Proton extrusion was inhibited by vanadate, suggesting the involvement of the plasma membrane H a ATPase in cell responses to salinity. There was virtually no eAect of NaCl on the net Ca 2a flux in protoplasts, while in the tissue a large transient Ca 2a eCux followed the salt treatment. Salt-induced Ca 2a eCux was essentially independent of external Ca2a concentrations in the range 0. 1t o 10 mM. Also, Ca2a flux responses were ‘saturated’ above 50 mM NaCl. It is suggested that almost all the measured Ca2a flux originates from Naa/Ca2a and Ha/Ca2a ion exchange in the cell wall. This conclusion was supported by the results of modelling cation exchange in the cell wall. # 2000 Annals of Botany Company

Noninvasive, ion-selective vibrating microelectrodes were used to measure the kinetics of H+, Ca2+, K+, and Cl− fluxes and the changes in their concentrations caused by illumination near the mesophyll and attached epidermis of bean (Vicia fabaL.). These flux measurements were related to light-induced changes in the plasma membrane potential. The influx of Ca2+ was the main depolarizing agent in electrical responses to light in the mesophyll. Changes in the net fluxes of H+, K+, and Cl− occurred only after a significant delay of about 2 min, whereas light-stimulated influx of Ca2+ began within the time resolution of our measurements (5 s). In the absence of H+ flux, light caused an initial quick rise of external pH near the mesophyll and epidermal tissues. In the mesophyll this fast alkalinization was followed by slower, oscillatory pH changes (5–15 min); in the epidermis the external pH increased steadily and reached a plateau 3 min later. We explain the initial alkalinization of the medium as a result of CO2 uptake by photosynthesizing tissue, whereas activation of the plasma membrane H+ pump occurred 1.5 to 2 min later. The epidermal layer seems to be a substantial barrier for ion fluxes but not for CO2diffusion into the leaf.

Non-invasive microelectrode flux measurement is a convenient tool to study membrane transport processes in plants in situ. Its high resolution (a few microns in space and several seconds in time) enables real-time studies of membrane transport processes at various levels of plant structural organization, from intact organs to various tissues, single cells or protoplasts derived from these cells. Being noninvasive, the technique allows flux measurements over several hours or days, even from rapidly moving tissues such as growing or nutating plant axial organs. Importantly, fluxes of several ions and neutral molecules can be measured concurrently, enabling not only quantitative estimates of rates of measured processes, but also providing some valuable information about the stoichiometry between activities of various membrane transporters and their time dependence. In this review, we revise the principles of non-invasive ion and neutral molecule flux measurements using the MIFE™ technique and discuss some methodological aspects of manufacturing, calibrating, and using flux measuring microelectrodes.

International audience; Non-invasive microelectrode ion flux measuring (the MIFE system) allows concurrent quantification of net fluxes of several ions with high spatial (several ?m) and temporal (ca 5 s) resolution. Over the last 10 years, the MIFE system has been widely used to study various aspects of salt stress signaling and adaptation in plants. This chapter summarizes some major findings in the area such as using MIFE for deciphering the specific and non-specific components of salinity stress, resolving the role of the plasma membrane H(+)-pump in salinity responses, proving K(+) homeostasis as a key feature of salinity tolerance, and discovering the mechanisms behind the ameliorative effects of Ca(2+) and other mitigating factors (such as polyamines or compatible solutes). The full protocols for microelectrode fabrication, calibration, and use are then given, and two basic routines for measuring net K(+) and Na(+) fluxes from salinity stressed roots are described in the context of plant screening for salt stress tolerance.

Growth and early gravitropic responses of corn roots in solution have been studied using time-lapse photography. Aeration was required for both root growth and gravitropism. The optimum pH for gravitropism was in the range 5 to 6. The bending response seemed to be greater for roots in non-buffered solution than in buffered solution. Fastest growth and maximum curvature occurred with about 0.2 mol m−3 Ca2+. Under some conditions, the gravitropic response started with apparently negligible time delay after the start of the gravitropic stimulus. This may denote graviperception in or near the elongation zone itself. This mechanism for early but relatively weak gravitropism may help to explain a variety of gravitropic responses such as the ‘early wrong way’ curvature, and the behaviour of roots whose columella cells lack amyloplasts. More rapid bending appears to start at about 20 min, which is consistent with observations on roots in humid air and with the accepted statolith model of perception in the root cap.

This paper examines the ionic composition of wound-induced electrical currents in higher plant tissue, using two non-injurious electrophysiological techniques. By simultaneous recording of K + , H + , and Ca2+ ion fluxes with extracellular ion-selective microelectrodes, we have determined that a Ca2+ influx (2-4 fzA cm-2), a small H+ influx (017 /zA cm"2) and a large K+ efflux (16 ft A cm"2) occur immediately after wounding in roots of Pisum sativum L. var. Greenfeast. Using an extracellular vibrating probe at the wound site, net ion currents of 26 fiA cm-2 were measured 5 min after wounding. In a more concentrated bathing medium (1/4 rather than 1/16 strength Hoagland's solution), net ion currents of 59 ftA cm 2 were measured, and these would appear to be the largest extracellular currents that have been measured in plants. We made a quantitative comparison of the summed ion fluxes with the net ion currents and this revealed that ion fluxes, in addition to those measured here, occur after wounding.

An account is given of an investigation of net ionic currents and specific ion fluxes occuring during the initiation of organogenesis in detached leaves of Graptopetalum paraguayense E. Walther, in which a dramatic change in growth polarity is cytomorphologically evident 3–5 d after leaf detachment from the plant. Using the vibrating probe, it was possible to identify a peak of ionic current which is focused over the area of the leaf base where organogenesis is initiated. This net current is largest during the initial 12h after leaf detachment. With ion-selective microelectrodes capable of measuring H+, K+ and Ca2+ ion fluxes simultaneously in the same region of the leaf base, H+ and K+ fluxes remain relatively steady during the initial 24 h after detachment, while a large lanthanum-sensitive Ca2+ influx decreases by 50% from 2 to 12h. By 24h, Ca2+ transport is dominated by an efflux. We present evidence from a quantitative comparison of the ion current data collected using these two techniques, that Ca2+, H+ and K+ transport accounts for the major electrogenic ion fluxes during 2 and 12 but not 24 h after leaf detachment. The possibility is addressed that these ion currents, which precede organogenesis, and in particular the predominant Ca2+ flux, play a role in the establishment of growth polarity in higher plant tissues.

Plant salinity tolerance is a polygenic trait with contributions from genetic, developmental, and physiological interactions, in addition to interactions between the plant and its environment. In this study, we show that in salt-tolerant genotypes of barley (Hordeum vulgare), multiple mechanisms are well combined to withstand saline conditions. These mechanisms include: (1) better control of membrane voltage so retaining a more negative membrane potential; (2) intrinsically higher H+ pump activity; (3) better ability of root cells to pump Na+ from the cytosol to the external medium; and (4) higher sensitivity to supplemental Ca2+. At the same time, no significant difference was found between contrasting cultivars in their unidirectional 22Na+ influx or in the density and voltage dependence of depolarization-activated outward-rectifying K+ channels. Overall, our results are consistent with the idea of the cytosolic K+-to-Na+ ratio being a key determinant of plant salinity tolerance, and suggest multiple pathways of controlling that important feature in salt-tolerant plants.

Ion flux kinetics associated with blue light (BL) treatment of two wild types (WTs) and the phot1 , phot2 and phot1/phot2 mutants of Arabidopsis were studied by using the MIFE noninvasive ion-selective microelectrode technique. BL induced significant changes in activity of H + and Ca 2+ transporters within the first 10 min of BL onset, peaking between 3 and 5 min. In all WT plants and in phot2 mutants, BL induced immediate Ca 2+ influx. In phot1 and phot1/phot2 mutants, net Ca 2+ flux remained steady. It is suggested that PHOT1 regulates Ca 2+ uptake into the cytoplasm from the apoplast. Changes in ion fluxes were measured from cotyledons of intact seedlings and from the cut top of the hypocotyl of decapitated seedlings. Thus the photoreceptors mediating BL-induced Ca 2+ and H + fluxes are present in the rest of the decapitated seedling and probably in the cotyledons as well. The H + and Ca 2+ flux responses to BL appear not to be linked because, ( i ) when changes were observed for both ions, Ca 2+ flux changed almost immediately, whereas H + flux lagged by about 1.5 min; ( ii ) in the Wassilewskija ecotype, changes in H + fluxes were small. Finally, wave-like changes in Ca 2+ and H + concentrations were observed along the cotyledon–hook axis regardless of its orientation to the light.

The transport of mineral ions into and out of tissues and cells is central to the life of plants. Ion transport and the plasma membrane transporters themselves have been studied using a variety of techniques. In the last 15 years, measurement of specific ion fluxes has contributed to the characterization of transport systems. Progress in molecular genetics is allowing gene identification and controlled expression of transporter molecules. However the molecular expression of transporter gene products must be characterized at the functional level. The ion-selective microelectrode technique to measure specific ion fluxes non-invasively is ideally suited to this purpose. This technique, its theory, its links with others and its application and prospects in plant science, are discussed. Ions studied include hydrogen, potassium, sodium, ammonium, calcium, chloride and nitrate. Applications discussed include: solute ion uptake by roots; gravitropism and other processes in the root cap, meristematic and elongation zones; Nod factor effect on root hairs; osmotic and salt stresses; oscillations; the effects of light and temperature. Studies have included intact roots, leaf mesophyll and other tissues, protoplasts and bacterial biofilms. A multi-ion capability of the technique will greatly assist functional genomics, particularly when coupled with imaging techniques, patch clamping and the use of suitable mutants.

Recent evidence suggests that in plants, as in vertebrates, natriuretic peptides (NPs) regulate homeostasis. In this study noninvasive ion-selective vibrating microelectrodes were used to measure net fluxes of K+, Na+, and H+ in Zea mays root conductive tissue. Immunoreactant plant natriuretic peptides (irPNP) cause immediate net H+ influx and delayed net K+ and Na+ uptake. Delayed net K+ influx was also observed in response to 8-Br-cGMP, however, not accompanied by significant changes in net H+ fluxes. Furthermore, 8-Br-cGMP does not stimulate the plasma membrane H+-ATPase implying that cGMP directly affects cation channels. The data are consistent with NP and cGMP-dependent stimulation of nonselective cation channels with P(K) > P(Na) and point to a complex role for NPs in plant homeostasis.

Seedling roots of corn were treated with different concentrations of mannitol-containing solution for 1 to 1.5 hr, and net fluxes of Ca2+ and H+ were measured in the elongation region. H+ fluxes were much more sensitive to osmotic pressure than were Ca2+ fluxes. Oscillations of 7-min period in H+ flux, normally observed in the control, were almost fully suppressed at high osmotic concentrations. Net H+ flux was shifted from average efflux of 25 +/- 3 nmol m-2 sec-1 to average influx of 10 +/- 5 nmol m-2 sec-1 after the incubation in 100 mm mannitol. The larger the osmotic concentration, the larger was the H+ influx. This flux caused the unbuffered solution of pH 4.85 to change to pH 5.3 after mannitol application. It appears that the osmoticum suppresses oscillatory H+ extrusion at the plasma membrane. Discrete Fourier Transforms of the H+ flux data showed that, apart from suppression of the 7-min oscillations in H+ flux, mannitol also promoted the appearance of faster 2-min oscillations. Ca2+ influx slightly increased after mannitol treatment. In addition the 7-min oscillatory component of Ca2+ flux remained apparent thereby showing independence of H+ flux.

Elongation growth of plant cells occurs by stretching of cell walls under turgor pressure when intermolecular bonds in the walls are temporarily loosened. The acid-growth theory predicts that wall loosening is the result of wall acidification because treatments (including IAA and fusicoccin) that cause lowered wall pH cause elongation. However, conclusive evidence that IAA primarily reduces wall pH has been lacking. Calcium has been reported to stiffen the cell walls. We have used a microelectrode ion-flux measuring technique to observe directly, and non-invasively, the net fluxes of protons and calcium from split coleoptiles of oats (Avena sativa L.) in unbuffered solution. Normal net fluxes are 10 nmol · m(-2) · s(-1) proton efflux and zero calcium flux. The toxin fusicoccin (1 μM) causes immediate efflux from tissue not only of protons, but also of calcium, about 110 nmol · m(-2) · s(-1) in each case. The data fit the "weak acid Donnan Manning" model for ion exchange in the cell wall. Thus we associate the known "acid-growth" effect of fusicoccin with the displacement of calcium from the wall by exchange for protons extruded from the cytoplasm. Application of 10 μM IAA causes proton efflux to increase transiently by about 15 nmol · m(-2) · s(-1) with a lag of about 10 min. The calcium influx decreases immediately to an efflux of about 20 nmol · m(-2) · s(-1). It appears that auxin too causes an "acid-growth" effect, with extruded protons exchanging for calcium in the cell walls.

K+, Ca2+ and H+ flux profiles were studied around the surface of germinating wheat (Triticum aestivum L. cv. Machete) seeds using a non-invasive microelectrode technique. The seed surface was scanned every 500 µm along the longitudinal axis, and net ion fluxes were measured 2 min and 1, 4 and 24 h after seed rehydration com-menced. Functionally different seed zones exhibited different ion exchange patterns, which also changed differently with time. One h after seed rehydration, the peak of H+ extrusion was observed in the embryo-scutellum region. By 24 h this peak had shifted towards the region of radicle emergence, and its magnitude had increased 5-fold. By con-trast, H+ activity in the endosperm region decreased noticeably during this period. At the same time, Ca2+ flux shifted from initial net influx to efflux in the embryo and scutellum zones. Most striking were K+ flux profiles, with large K+ efflux observed at each end and almost zero flux near the geometrical center of the seed. Significant K+ extru-sion from the embryo end was observed for as long as 24 h after seed rehydration and was not related to the loss of membrane integrity. Seed treatment with metabolic inhibitors (vanadate and tetraethylammonium) caused signifi-cant changes in both H+ and K+ fluxes suggesting that at least part of the measured fluxes originated from activities of plasma membrane transporters. We discuss possible mechanisms responsible for the observed regularities and question whether the seed coat in wheat caryopses is as ion-impermeable as previously believed.

Verapamil application in ion transport studies on plant cells is widespread; however, the mechanism of its action is still poorly understood. Net flux of Ca 2+ , K + , Na + , H + and Cl – were measured in solution around oat seedlings using the non-invasive ion-selective microelectrode MIFE technique. The verapamil effect on intact plant tissues was a distinct immediate influx of monovalent cations, H + , K + or Na + . Pre-treatment with tetraethyl-ammonium, Cs + , Ba 2+ or Ca 2+ did not affect K + flux changes, whereas DMSO, Cd 2+ and vanadate increased verapamil-induced K + influx. Verapamil-induced K + flux increased with increased external KCl concentration or pH. Verapamil concentration up to 1 mM failed to shift Ca 2+ flux to efflux. Pre-treatment with adrenaline and dopamine agonists and antagonists led to changes in verapamil-induced ion flux, especially for Ca 2+ . We suggest that a transporter that allows entry of K + and Na + was the main system providing monovalent ion influx after verapamil application.

The electric potential difference changes observed on etiolated oat coleoptiles in response to phytochrome transformation have been further studied using contacts on the coleoptile surface. Results are given, at 0.4 second resolution, for the first 1.5 minutes after saturating flashes of light each lasting 1 second. Responses to initial red (662 nanometers), to far red (about 700 nanometers and above) 10 minutes later, and to second red 10 minutes later still, all have time courses that are approximately Gaussian sigmoid in shape. The response to far red is of opposite sign to the response to red. Approximate magnitudes of the three changes, 1 minute after the light flash, are +6 millivolts for red, −10 millivolts for far red, +3.5 millivolts for the second red. It is argued that the observations reflect a hyperpolarization of the plasmalemma of coleoptile cells following red light and depolarization following far red. The response to red is not produced by a change in membrane permeability to K + . The mechanism could include a change to Na + or Cl − permeability or a modulation of an electrogenic pump: enhanced H + extrusion, Ca 2+ extrusion, or Cl − uptake. The response to far red could be produced by the reverse of one of those changes. The Gaussian curve is fitted to the data to determine the time at which the responses begin. Each response begins 4.5 seconds after the start of the flash of light. These delays are not related to the time course of phytochrome pelletability or redistribution in the cell. The delays may be due to some interaction of the transformed phytochrome with the plasma-lemma. Alternatively, the transformed phytochrome may interact quickly with some other structure which initiates a signal that takes 4.5 seconds to reach the plasmalemma.

Brief exposures to red light induce far red-reversible changes of 5 to 10 millivolts magnitude in the upper 1 centimeter of etiolated Avena coleoptiles. The changes begin within 15 seconds of the start of illumination and they continue for at least 12 minutes. The changes were measured using a flowing solution of 10 mm KCl to contact the surface of the coleoptile. A dark-grown coleoptile shows no change in response to far red light unless it first receives red light treatment. The second of two red light exposures is ineffective without an intervening far red treatment. Some characterization of these electric responses to light is presented.

The H(+) cotransport of neutral and acidic amino acids induces transient depolarizations of oat coleoptile (Avena sativa L., var Victory) plasma membranes. The depolarizations, which are completed within 1 or 2 minutes, are followed by repolarizations that are nearly completed within another 2 or 3 minutes. Cysteine induced a two-phased alkalinization of the tissue free space during the electrical changes. The first phase was a rapid, linear increase in pH that coincided with the depolarization; the second phase was a slower, also linear, increase in pH that coincided with the repolarization. Reacidification did not occur until cysteine was withdrawn. Five other acidic, basic, and neutral amino acids also induced persistent alkalinization of the free space.The notable features of these measurements are that free-space pH was measured more directly than previously, that pH changes corresponded in time to the electrical potential changes, and that reacidification of the free space did not occur. The latter observation indicates that net H(+) efflux did not occur during repolarization and that the repolarizing current was carried by some other ion. We propose that repolarization could have depended upon depolarization-induced changes in passive K(+) fluxes combined with an enhanced H(+) extrusion that increased until it equaled, but did not exceed, the enhanced influx of H(+).In support of the feasibility of our hypothesis, we present a quantitative simulation model for cotransport. The simulation model also provides an interpretation of the unique electrical effects of histidine and the basic amino acids. In addition, the model focuses attention upon the difficulties of interpreting H(+)-anion cotransport.