Potassium transport in the rabbit renal proximal tubule: Effects of barium,ouabain, valinomycin,and other ionophores

Summary Potassium fluxes in a suspension of rabbit proximal tubules were monitored using a potassium-sensitive extracellular electrode. Ouabain (10^–4 m) and barium (5mm) were used to selectively quantitate the potassium efflux pathway (105±5 nmol K⁺·mg protein^–1·min^–1) and the sodium pump-related potassium influx (108±7), respectively. These equal and opposite fluxes suggest that potassium accumulation in the cell occurs mainly through the sodium pump and that potassium efflux occurs mainly through barium-sensitive potassium channels. Thus the activity of the sodium pump (Na, K-ATPase) in the basolateral membrane of the proximal tubule is balanced by the efflux of potassium, presumably across the basolateral membrane, which has a high potassium permeability. In addition, the effect of valinomycin and other ionophores was examined on potassium fluxes and several metabolic parameters [oxygen consumption (QO₂), ATP content]. The addition of valinomycin to the tubules produced a net efflux of potassium which was quantitatively equivalent to the efflux produced by the addition of ouabain. The valinomycin-induced efflux was mainly due to the activity of valinomycin as a mitochondrial uncoupler, which indirectly inhibited the sodium pump by allowing a rapid reduction of the intracellular ATP. Amphotericin, nystatin, and monensin all produced large net releases of intracellular potassium. The action of the ionophores could be localized to the plasma or mitochondrial membrane and classified into three groups, as follows: (a) those which demonstrated full mitochondrial uncoupler activity (FCCP, valinomycin), (b) those which had no uncoupler activity (amphotericin B, nystatin); and (c) those which displayed partial uncoupler activity (monensin, nigericin).     

Molecular basis of ion permeability in a voltage‐gated sodium channel

Voltage‐gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na⁺ ≈ Li⁺ ≫ K⁺, Ca²⁺, Mg²⁺) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.     

Transport of alkali cations through thin lipid membranes by (222)C10-Cryptand,an ionizable mobile carrier

Summary The kinetics of K⁺ and Na⁺ transport across the membrane of large unilamellar vesicles (L.U.V.) were compared at two pH's, with two carriers: (222)C ₁₀-cryptand (diaza-1, 10-decyl-5-hexaoxa-4,7,13,16,21,24-bicyclo[8.8.8.]hexacosane) and valinomcyin, i.e. an ionizable macrobicyclic amino polyether and a neutral macrocyclic antibiotic. The rate of cation transport by (222)C₁₀ saturated as cation and carrier concentrations rose. The apparent affinity of (222)C₁₀ for K⁺ was higher and less pH dependent than that for Na⁺ but resembled the affinity of valinomycin for K⁺. The efficiency of (222)C₁₀ transport of K⁺ decreased as the pH fell and the carrier concentration rose, and was about ten times lower than that of valinomycin. Noncompetitive K⁺/Na⁺ transport selectivity of (222)C₁₀ decreased as pH, and cation and carrier concentrations rose, and was lower than that of valinomycin. Transport of alkali cations by (222)C₁₀ and valinomycin was noncooperative. Reaction orders in cationn(S) and carrierm(M) varied with the type of cation and carrier and were almost independent of pH;n(S) andm(M) were not respectively dependent on carrier or cation concentrations. The apparent estimated constants for cation translocation by (222)C₁₀ were higher in the presence of Na⁺ than of K⁺ due to higher carrier saturation by K⁺, and decreased as pH and carrier concentration increased. Equilibrium potential was independent of the nature of carrier and transported cation. Results are discussed in terms of the structural, physicochemical and electrical characteristics of carriers and complexes.     

The Rate Constants of Valinomycin-Mediated Ion Transport through Thin Lipid Membranes

Electrical relaxation experiments have been performed with phosphatidylinositol bilayer membranes in the presence of the ion carrier valinomycin. After a sudden change of the voltage a relaxation of the membrane current with a time constant of about 20 μsec is observed. Together with previous stationary conductance data, the relaxation amplitude and the relaxation time are used to evaluate the rate constants of valinomycin-mediated potassium transport across the lipid membrane. It is found that the rate constants of translocation of the free carrier S and the carrier-ion complex MS⁺ are nearly equal (2·10⁴ sec^-1) and are of the same order as the dissociation rate constant of MS⁺ in the membrane-solution interface (5·10⁴ sec^-1). The equilibrium constant of the heterogeneous association reaction M⁺ (solution) + S (membrane) → MS⁺ (membrane) is found to be ~ 1 M^-1, about 10⁶ times smaller than the association constant in ethanolic solution.     

Alkali ion transport through lipid bilayer membranes mediated by enniatin A and B and beauvericin

Summary Stationary conductance measurements with lipid bilayer membranes in the presence of enniatin A and B and beauvericin were performed. For comparison, some valinomycin systems were investigated. It was found that the conductance in the case of enniatin A and B is caused by a carrier ion complex with a 11 stoichiometry, whereas for beauvericin, a 31 carrier ion complex has to be assumed to explain the dependence of the conductance on carrier and ion concentration in the aqueous phase. The current-voltage curves measured with dioleoyl phosphatidylcholine membranes show a superlinear behavior for the three carriers in the presence of potassium. On the other hand, supralinear current-voltage curves were observed with membranes from different monoglycerides, except for beauvericin. The results obtained with enniatin A and B are in a satisfactory agreement with an earlier proposed carrier model assuming a complexation between carrier and ion at the membrane water interface.The discrimination between potassium and sodium ions is much smaller for the enniatins than for valinomycin. This smaller selectivity as well as the fact that potassium ions cause the highest conductance with lipid bilayer membranes may be due to the smaller size of the cyclic enniatin molecules, which contain 6 residues in the ringvs. 12 in the case of valinomycin. Charge-pulse relaxation studies were performed with enniatin A and B, beauvericin, and valinomycin. For monoolein membranes only in the case of valinomycin, all three relaxations predicted by the model could be resolved. In the case of the probably more fluid membranes from monolinolein (^{9, 12}-C_{18: 2}) and monolinolenin (^{9, 12, 15}-C_{18: 3}) for all carrier systems except for beauvericin, three relaxations were observed.The association rate constantk _R , the dissociation rate constantk _D , and the two translocation rate constantsk _MS andk _s for complexed and free carrier, respectively, could be calculated from the relaxation data. The carrier concentration in the aqueous phase had no influence on the rate constants in all cases, whereas a strong saturation of the association rate constantk _R with increasing ion concentration was found for the enniatins. Because of the saturation,k _R did not exceed a value of 4×10⁵ m ^–1 sec^–1 with 1m salt irrespective of carrier, ion, or membrane-forming lipid.A similar but less pronounced saturation behavior was also observed for the translocation rate constantk _S of the free carrier. The other two rate constants were independent of the ion concentration in the aqueous phase. In the case of the enniatins, the translocation rate constantk _MS was not independent from the kind of the transported ion. In the series K⁺, Rb⁺ and Cs⁺,k _MS increases about threefold. The turnover numbers for the carriers as calculated from the rate constants range between 10⁴ sec^–1 and 10⁵ sec^–1 and do not show a strong difference between the individual carriers. The conductance difference in the systems investigated here is therefore mainly caused by the partition coefficients, which are smaller for the enniatins than for valinomycin.     

Some effects of trinitrocresolate and valinomycin on Na and K transport across thin lipid bilayer membranes: A steady-state analysis with simultaneous tracer and electrical measurements

Summary This paper describes the effect of trinitrocresolate anions (TNC^–) on the electrical conductance (G _m ), and tracer-measured unidirectional Na and K fluxes (M _Na andM _K) across bilayers formed from sheep red cell lipids dissolved in decane. In the absence of TNC^–, typical low conductances were observed, while the cation fluxes were too low to measure by our techniques (<10^–12 moles cm^–2 sec^–1). In the presence of TNC^– (10^–2 m),G _m increased and TNC^– was the main charge carrier in the system. The cationic fluxes were also much increased, but the membranes showed no significant selectivity between K and Na. Furthermore, the Na and K fluxes were at least two orders of magnitude larger than the ionic fluxes calculated fromG _m . Thus, almost all of the K and Na transport across the membrane in the presence of TNC^– is electrically silent and is probably carried out as KTNC and NaTNC ion pairs.In the presence of valinomycin (10^–6 m) and no TNC^–, both the ion fluxes andG _m were 10³ times larger in KCl than in NaCl, thus exhibiting the characteristic high selectivity of valinomycin for K over Na. In the presence of both valinomycin (10^–6 m) and TNC^– (10^–2 m), this selectivity disappeared in that bothG _m andM _Na in the NaCl system were similar to the respective values in the KCl system. Even under these conditions, most of the Na is still transported by a process which does not carry charge.BothG _m andM _x increased alike and monotonically with increasing temperature over the range 7 to 30°C. In the absence of TNC^– the enthalpies of activation were invariably higher in KCl than in NaCl. Addition of TNC^– produced equal enthalpies of activation for both Na and K containing systems suggesting a common, temperature-dependent, ratedetermining step in charge transfer and the electrically silent cation fluxes.     

Cation permeability ratios of sodium channels in normal and grayanotoxin-treated squid axon membranes

Summary Permeabilities of squid axon membranes to various cations at rest and during activity have been measured by voltage clamp before and during internal perfusion of 4×10^–5 m grayanotoxin I. The resting sodium and potassium permeabilities were estimated to be 6.85×10^–8 cm/sec and 2.84×10^–6 cm/sec, respectively. Grayanotoxin I increased the resting sodium permeability to 7.38×10^–7 cm/sec representing an 11-fold increase. The potassium permeability was increased only by a factor of 1.24. The resting permeability ratios as estimated by the voltage clamp method before application of grayanotoxin I were Na (1): Li (0.83): formamidine (1.34): guanidine (1.49): Cs (0.87): methylguanidine (0.86): methylamine (0.78). Grayanotoxin I did not drastically change the resting permeability ratios with a result of Na (1): Li (0.95): formamidine (1.27): guanidine (1.16): Cs (0.47): methylguanidine (0.72): methylamine (0.46). The membrane potential method gave essentially the same resting permeability ratios before and during application of grayanotoxin I if corrections were made for permeability to choline as the cation substitute and for changes in potassium permeability caused by test cations. The permeability ratio choline/Na was estimated to be 0.72 by the voltage clamp method and 0.65 by the membrane potential method. Grayanotoxin I decreased the ratio to 0.43. The permeability ratios during peak transient current were estimated to be Na (1): Li (1.12): formamidine (0.20): guanidine (0.20): Cs (0.085): methylguanidine (0.061): methylamine (0.036). Thus the sodium channels for the peak current are much more selective to cations than the resting sodium channels. It appears that the resting sodium channels in normal and grayanotoxin I-treated axons are operationally different from the sodium channels that undergo a conductance increase upon stimulation.     

Cation Activation of the Basolateral Sodium-Potassium Pump in Turtle Colon

The current generated by electrogenic sodium-potassium exchange at the basolateral membrane of the turtle colon can be measured directly in tissues that have been treated with serosal barium (to block the basolateral potassium conductance) and mucosal amphotericin B (to reduce the cation selectivity of the apical membrane). We studied the activation of this pump current by mucosal sodium and serosal potassium, rubidium, cesium, and ammonium. The kinetics of sodium activation were consistent with binding to three independent sites on the cytoplasmic side of the pump. The pump was not activated by cellular lithium ions. The kinetics of serosal cation activation were consistent with binding to two independent sites with the selectivity Rb > K > Cs > NH₄. The properties and kinetics of the basolateral Na/K pump in the turtle colon are at least qualitatively similar to those ofthe well-characterized Na/K-ATPase of the human red blood cell .     

THE ELECTRICAL CONDUCTIVITY OF SODIUM AND POTASSIUM GUAIACOLATES IN GUAIACOL

The data of the author and Uhlig, and new data, on the conductivity of sodium and of potassium guaiacolates in guaiacol at 25° have been computed with an improved conductance equation which is valid to somewhat higher concentrations than the equations formerly used. The new constants are, Λ₀ = 9.0, K = 2.8 x 10^–5 for sodium guaiacolate and Λ₀ = 9.5, K = 3.4 x 10^–5 for potassium guaiacolate.     

A circular dichroism study of valinomycin barium ion complex

Complex formation of valinomycin with Ba²⁺ ions was investigated by circular dichroism spectroscopy. The results indicated that Ba²⁺ forms entirely different types of complexes when compared with K⁺. The data with perchlorate salt showed evidence for the formation of less stable V₂C (peptide sandwich), VC (1:1), and VC₂ (ion sandwich) complexes followed by a stable final complex upon gradual addition of salt (V stands for valinomycin and C for the cation). This final complex possibly has a flat structure with no internal hydrogen bonds, similar to that of valinomycin in highly polar solvents. The possible complexation mechanism and the role played by anions and isopropyl side chains are highlighted.     

Thermodynamics of ATP hydrolysis from membrane electrode measurements of metal-ion ATP and ADP complexation

Free energies for the ATP-ADP hydrolysis reaction have been recalculated on the basis of new thermodynamic formation constants for metal-ion ATP and ADP complexes as determined by potentiometric measurements with ion-selective membrane electrodes. It is shown that free-energy maps are sharply altered at metal-ion levels greater than 10^?3m because of the effect of previously unrecognized 2:1 complexes of divalent metal ions with ATP and ADP. New formation constants for ADP complexes with sodium, potassium, calcium, and magnesium are also reported.     

ON GUAIACOL SOLUTIONS : I. THE ELECTRICAL CONDUCTIVITY OF SODIUM AND POTASSIUM GUAIACOLATES IN GUAIACOL

1. Measurements on the densities, viscosities, dielectric constants, and specific conductances of pure anhydrous and water-saturated guaiacol at 25°C. are reported. 2. The solubility of water in guaiacol at 25°C., and its effect on the electrical conductivity of a sodium guaiacolate solution is given. 3. Electrical conductivity measurements are reported on solutions of sodium and potassium guaiacolates in water-saturated guaiacol at 25°C. 4. The decrease of electrical conductivity with increasing concentration for these salts is explained on the basis of an ionic equilibrium combined with the interionic attraction theory of Debye and Hückel. 5. The limiting equivalent conductances of sodium and potassium guaiacolates in water-saturated guaiacol at 25°C., the corresponding limiting ionic mobilities, and the dissociation constants are computed from the conductivity measurements. The salts are found to be weak electrolytes with dissociation constants of the order of 5 x 10^–6.     

Potassium accumulation in growing Methanobacterium thermoautotrophicum and its relation to the electrochemical proton gradient

Cultures of Methanobacterium thermoautotrophicum (Marburg) growing on media low in potassium accumulated the cation up to a maximal concentration gradient ([K⁺]_{intracellular}/[K⁺]_{extracellular}) of approximately 50,000-fold. Under these conditions, the membrane potential was determined by measuring the equilibrium distribution of the lipophilic cation (¹⁴C) tetraphenylphosphonium (TPP⁺). This cation was accumulated by the cells 350-to 1,000-fold corresponding to a membrane potential (inside negative) of 170–200 mV. The pH gradient, as measured by equilibrium distribution of the weak acid, benzoic acid, was found to be lower than 0.1 pH units (extracellular pH=6.8). The addition of valinomycin (0.5–1 nmol/mg cells) to the culture reduced the maximal concentration gradient of potassium from 50,000-to approximately 500-fold, without changing the membrane potential. After dissipation of the membrane potential by the addition of ¹²C-TTP⁺ (2 mol/mg cells) or tetrachlorosalicylanilide (3 nmol/mg cells), a rapid and complete efflux of potassium was observed.These data indicate that potassium accumulation in the absence of valinomycin is not in equilibrium with the membrane potential. It is concluded that at low extracellular K⁺ concentrations potassium is not accumulated by M. thermoautotrophicum via an electrogenic uniport mechanism.Non-common abbreviations TPP⁺ Tetra phenylphosphonium bromide - DTE Dithioerythritol - TCS 3,5,3,4-Tetrachlorosalycylanilide     

Conserved Aromatic Residue Confers Cation Selectivity in Claudin-2 and Claudin-10b

In tight junctions, both claudin-2 and claudin-10b form paracellular cation-selective pores by the interaction of the first ECL 1 with permeating ions. We hypothesized that a highly conserved aromatic residue near the pore selectivity filter of claudins contributes to cation selectivity by cation-π interaction with the permeating cation. To test this, we generated MDCK I Tet-off cells stably transfected with claudin-2 Tyr⁶⁷ mutants. The Y67L mutant showed reduced cation selectivity compared with wild-type claudin-2 due to a decrease in Na⁺ permeability, without affecting the Cl⁻ permeability. The Y67A mutant enlarged the pore size and further decreased the charge selectivity due to an increase in Cl⁻ permeability. The Y67F mutant restored the Na⁺ permeability, Cl⁻ permeability, and pore size back to wild-type. The accessibility of Y67C to methanethiosulfonate modification indicated that its side chain faces the lumen of the pore. In claudin-10b, the F66L mutant reduced cation selectivity, and the F66A mutant lost pore conductance. We conclude that the conserved aromatic residue near the cation pore domain of claudins contributes to cation selectivity by a dual role of cation-π interaction and a luminal steric effect. Our findings provide new insight into how ion selectivity is achieved in the paracellular pore.     

1-Anilino-8-naphthalenesulfonate: A fluorescent probe of ion and ionophore transport kinetics and trans-membrane asymmetry

Summary The kinetics of the transport of the 1-anilino-8-naphthalenesulfonate (ANS^–, an anionic fluorescent probe of the membrane surface) across phospholipid vesicle membranes have been studied using a stopped-flow rapid kinetic technique. The method has been used to gain detailed information about the mechanism of transport of this probe and to study ionophore-mediated cation transport across the membrane. The technique has also been exploited to study differences between the inside and outside surfaces of vesicles containing phosphatidyl choline (PC).The following is a summary of the major conclusions of this study. (a) Binding of ANS^– on the outside surface occurs within times shorter than 100 sec while permeation occurs in the time range 5–100 sec. (b) Net transport of ANS^– occurs with cotransport of alkali cations. (c) The transport rate is maximal in the region of the crystalline to liquidcrystalline phase transition, and the increase correlates with changes in the degree of aggregation of the vesicles. (d) Incorporation of phosphatidic acid (PA), phosphatidyl ethanolamine (PE) or cholesterol into PC membranes decreases the rate of ANS^– transport. (e) Neutral ionophores (I) of the valinomycin type increase ANS^– permeability in the presence of alkali cations (M ⁺) by a mechanism involving the transport of a ternaryI–M ⁺-ANS^– complex. The equilibrium constants for formation of these complexes and their rate constants for their permeation are presented. The maximal turnover number for ANS^– transport by valinomycin in dimyristoyl PC vesicles at 35°C was 46 per sec. (f) The partitioning of the ionophore between the aqueous and membrane phases and the rate of transfer of an ionophore from one membrane have been determined in kinetic experiments. (g) A method is described for the detection ofI–M ⁺ complexes on the membrane surface by their enhancement effects on ANS^– fluorescence at temperature below the phase transition temperature on monolayer vesicles. The apparent stability constants for severalI–M ⁺ complexes are given. (h) Analysis of the effect of ionic strength on the ANS^– binding to the inside outside surfaces indicates that the electrostatic surface potential (at fixed ionic strength and surface change) is larger for the inside surface than for the outside surface. (i) Analysis of the dependence of the maximal ANS^– binding for the inside and outside surfaces of vesicles made from PC and a variable mole fraction of PA, PE or cholesterol indicate that the latter three are located preferentially on the inside surface.     

Respiration-independent Binding of SR to Bean Mitochondria

Binding of Sr²⁺ to bean mitochondria (Phaseolus vulgaris) shows a dissociation constant of 25 × 10⁻⁶ and results in 40 to 50 nmoles of Sr²⁺ bound per mg protein. The binding is partially inhibited by valinomycin plus K⁺, 2, 4-dinitrophenol, as well as ruthenium red at a level of the 120 nmoles per mg protein. These compounds also partially inhibit active uptake of Sr²⁺. Calcium and Mg²⁺ also partially inhibit binding in the same magnitude as previously reported for inhibition of transport. Phosphate which is required for divalent cation transport is without effect on the binding of Sr²⁺. The possible role of the observed binding sites in divalent cation transport is discussed.     

Coordinated cations in dipicolinato complexes of divalent metal ions

Several salts of alkali, alkaline earth metal and organic ammonium cations of a complex anion [ML₂]²⁻ {Where L = dipicolinato dianion, M = copper(II), nickel(II) and zinc(II)} are prepared. The coordination effect of [ML₂]²⁻ with the cations such as sodium, potassium, calcium, magnesium, and organic cations namely diammonium cation of 1,5-pentanediamine, diammonium cation of 1,8-octyldiamine, mono ammonium cation of 4-aminobenzylamine are studied by determining their X-ray crystal structures. Depending on the nature of cations, four different types of structures are obtained. When calcium is the cation a polymeric structure with calcium ions bridging the [ML₂]²⁻ is observed. The salts having sodium and potassium cations form polymeric chain like structures by oxo and aqua bridges. In the case of magnesium, the hydrated form of magnesium cations coordinates to [ML₂]²⁻. The organic ammonium salts of [ML₂]²⁻ have the structural features of conventional ionic complexes. These salts easily exchange cations. The organic ammonium salts of [ML₂]²⁻ decomposes to give the corresponding metal oxides at relatively low temperature range 300-450 °C.     

Stability and solvation of alkali metal ion complexes with dibenzo-30-crown-10

Stability constants for the 1:1 complexes of dibenzo-30-crown-10 (DB30C10) with alkali metal ions have been determined at 25 °C in nitromethane and water by conductometry and capillary electrophoresis, respectively. Transfer activity coefficients of DB30C10 and its complexes from nitromethane to S (S = water, acetonitrile, propylene carbonate, methanol, and N,N-dimethylformamide) have been determined at 25 °C to evaluate the solvation properties. The stability constant in the poorly solvating solvent, nitromethane, decreases with increasing metal ion size, Na⁺ > K⁺ > Rb⁺ > Cs⁺, reflecting the intrinsic selectivity governed by electrostatic interaction between the metal ion and the ether oxygen atoms. It is also suggested that a part of the ether oxygen atoms does not bind to the metal ion in the Na(DB30C10)⁺ complex. The aqueous stability constant varies as Na⁺ ? K⁺ ≈ Rb⁺ ≈ Cs⁺; this selectivity pattern is similar to that in acetonitrile, propylene carbonate, and methanol. The complex stability in water is very low compared to that in the nonaqueous solvents, owing to hydrogen bonding of water to the oxygen atoms of the free crown ether. The transfer activity coefficient values show that DB30C10 shields all the metal ions effectively from the solvents and lead to the conclusion that the complexation selectivity in S receives a significant contribution from the solvation of the free metal ions. The Na(DB30C10)⁺ complex has specific interaction with water, causing much lower K⁺/Na⁺ selectivity in H₂O than in MeOH.     

Human telomeric G-quadruplex DNA interactions of N-phenanthroline glycosylamine copper(II) complexes

We report in this article the interactions of five N-(1,10-phenanthrolin-5-yl)-β-glycopyranosylamine copper(II) complexes with G-quadruplex DNA. Specifically, the interactions of these compounds with a human telomeric oligonucleotide have been assessed by fluorescence-based assays (FRET melting and G4-FID), circular dichroism and competitive equilibrium dialysis experiments. The metal complexes bind and stabilize G-quadruplex DNA structures with apparent association constants in the order of 10⁴–10⁵ M⁻¹ and the affinity observed is dependent on the ionic conditions utilized and the specific nature of the carbohydrate moiety tethered to the 1,10-phenanthroline system. The compounds showed only a slight preference to bind G-quadruplex DNA over duplex DNA when the quadruplex DNA was folded in sodium ionic conditions. However, the binding affinity and selectivity, although modest, were notably increased when the G-quadruplex DNA was folded in the presence of potassium metal ions. Moreover, the study points towards a significant contribution of groove and/or loop binding in the recognition mode of quadruplex structures by these non-classical quadruplex ligands. The results reported herein highlight the potential and the versatility of carbohydrate bis-phenanthroline metal-complex conjugates to recognize G-quadruplex DNA structures.     

Size selectivity of aqueous pores in stomatous cuticles of <Emphasis Type="Italic">Vicia faba</Emphasis> leaves

Size selectivity of aqueous pores in Vicia leaf cuticles was investigated by measuring the penetration of calcium salts into the abaxial surface of detached leaves. Molecular weights of salts ranged from 111 g mol^–1 to 755 g mol^–1. Penetration in light at 20°C and 100% humidity was a first order process and rate constants of penetration ranged from 0.39 h^–1 (CaCl₂) to 0.058 h^–1 (Ca-lactobionate). Penetration was a first order process in the dark as well, but the rate constants were smaller by a factor of 1.82. Plotting logarithmatised rate constants versus anhydrous molecular weights resulted in straight lines both in light and in the dark. The slopes per hour were very similar and the average slope was –1.2×10^–3 mol g^–1. Hence, size selectivity was not affected by stomatal opening, and in light or darkness permeability of Vicia cuticles decreased by a factor of 2.9 when molecular weight increased from 100 g mol^–1 to 500 g mol^–1. Silver nitrate was preferentially precipitated as silver chloride in guard cells, glandular trichomes and at the base of trichomes. It was concluded that these precipitates mark the location of aqueous pores in Vicia leaf cuticles. The size selectivity of aqueous pores in Vicia leaf cuticles is small compared to that observed in poplar leaf cuticles, in which permeability decreased by a factor of 7–13 for the same range of molecular weights. It is also much smaller than size selectivity of the lipophilic pathway in cuticles. These findings suggest that active ingredients of pesticides, growth regulators and chemical inducers with high molecular weights penetrate leaves at higher rates when formulated as ions.