What is the basis of pederin polymorphism in Paederus riparius rove beetles? The endosymbiotic hypothesis

Paederus riparius (Coleoptera:Staphylinidae) females are polymorphic with regard to their ability to synthesize the hemolymph toxin pederin, a character confined to that sex: (+)-females endow their eggs with pederin whereas (–)-females lay eggs devoid of the substance. When reared without access to conspecifics, (+)-females can only be obtained from (+)-mothers. Ingestion of (+)-eggs during larval development, however, enables the offspring of (–)-females to also become (+)-females. This is only the case if untreated (+)-eggs are eaten, while sterilized (+)-eggs do not alter larval fate as (–)-females. The character can thus be transferred to unrelated specimens. These experiments suggest that microorganisms might be involved in the biosynthesis of this unusual compound. Pederin appears to be the first defensive substance in insects traceable to endosymbionts.     

Possible genetic basis of pederin polymorphism in rove beetles (Paederus riparius)

In Paederus riparius, (+) females able to biosynthesize the unique hemolymph toxin pederin and (-) females lacking this ability co-occur in natural populations. Larvae descended from both types of females were reared in the laboratory and the imagoes were crossed in order to get information about a possible genetic basis of this polymorphism. The daughters of (+) mothers become (+) females or (-) females, while the progeny of (-) mothers comprises only (-) females. This suggests a matrilineal trait because pederin biosynthesis cannot be inherited from the father. The rather stable proportion of nearly 90% (+) females in collected females is not maintained, however, when the beetles are reared in the laboratory. This observation is discussed with regard to artificial rearing conditions, where individuals are kept separate and cannot prey on conspecifics.     

Differential efficacy of toxic pederin in deterring potential arthropod predators of Paederus (Coleoptera: Staphylinidae) offspring

This study investigates the effects of pederin, a hemolymph toxin that is accumulated in the eggs of most Paederus females, on potential arthropod predators of the offspring of P. fuscipes and P. riparius. Insects generally do not respond to pederin present in the prey. Paederus larvae are sufficiently agile to escape from these predators by running away, and the eggs are hidden by the females. Unlike insects, (wolf) spiders are deterred by prey with pederin. They turn away from larvae they have already captured and exhibit cleansing behavior. Larvae containing pederin survive the attacks of spiders without damage, whereas larvae descended from females that do not transfer pederin into their eggs are often killed and eaten. In the case of sudden attacks by spiders, the larvae have no chance of escape. Their survival thus depends on chemical defense. These investigations show for the first time why pederin might be of considerable importance for Paederus in the field.     

Molecular identification of an endosymbiotic bacterium associated with pederin biosynthesis in Paederus sabaeus (Coleoptera: Staphylinidae)

Biosynthesis of the structurally complex hemolymph toxin pederin is an eminent character of Paederus females. For that capability, however, they rely on endosymbiotic bacteria that are lacking in aposymbiotic females. The bacterial inhabitants of the two phenotypes in Paederus sabaeus are evaluated in a PCR-based analysis of 16S rDNA. A certain fragment, which is not found in aposymbiotic females, is highly dominant in the other, biosynthesizing females and thus identifies the endosymbiont. Its DNA sequence reveals a member of the gamma subdivision of the Proteobacteria that is clustered within the genus Pseudomonas (sensu stricto) as it is most closely related to Pseudomonas aeruginosa. These bacteria appear as the hypothesized common producers of pederin and the pederin family of analogs from marine sponges.     

Evidence for a symbiosis island involved in horizontal acquisition of pederin biosynthetic capabilities by the bacterial symbiont of Paederus fuscipes beetles

Pederin belongs to a group of antitumor compounds found in terrestrial beetles and marine sponges. It is used by apparently all members of the rove beetle genera Paederus and Paederidus as a chemical defense against predators. However, a recent analysis of the putative pederin biosynthesis (ped) gene cluster strongly suggests that pederin is produced by bacterial symbionts. We have sequenced an extended region of the symbiont genome to gain further insight into the biology of this as-yet-unculturable bacterium and the evolution of pederin symbiosis. Our data indicate that the symbiont is a very close relative of Pseudomonas aeruginosa that has acquired several foreign genetic elements by horizontal gene transfer. Besides one functional tellurite resistance operon, the region contains a genomic island spanning 71.6 kb that harbors the putative pederin biosynthetic genes. Several decayed insertion sequence elements and the mosaic-like appearance of the island suggest that the acquisition of the ped symbiosis genes was followed by further insertions and rearrangements. A horizontal transfer of genes for the biosynthesis of protective substances could explain the widespread occurrence of pederin-type compounds in unrelated animals from diverse habitats.     

Effects of mass releases of Trichogramma brassicae on predatory insects in maize

Abstract Females of the rove beetle genus Paederus (Coleoptera: Staphylinidae) accumulate large amounts of the defensive compound pederin produced by an endosymbiotic bacterium. Pederin is transferred in the eggs, while the endosymbionts are transmitted via the egg shell. In all three species analyzed (P. melanurus, P. riparius, and P. sabaeus), descendants of aposymbiotic matrilines (lacking both endosymbionts and biosynthetic capabilities) acquire biosynthetic capability by the ingestion of eggs with endosymbionts during larval development. Successful colonization by endosymbionts depends on the number of eggs consumed and the age of the feeding larvae. During the adult stage, however, the females cannot acquire biosynthetic capabilities. Adult females are able to sequester pederin from eggs eaten, and they transfer the substance into their own eggs. Lack of intrinsic biosynthesis after ingestion of endosymbionts indicates that these are not biosynthetically active within the gut and have to reach an unknown internal location before completion of metamorphosis. These results are discussed with regard to the benefit of intraspecific cannibalism.     

Hybridization of chromosome-polymorphic populations of the inquiline ant,Doronomyrmex kutteri (Hym., Formicidae)

Summary The workerless, inquiline ant,Doronomyrmex kutteri has isolated populations with a haploid chromosome number ofn=23 both in the Alps (Swiss and South Tyrolean Alps) and in Sweden, and a population withn=25 in southern Germany. Crossbreeding of sexuals from all populations proved successful. Backcrosses of F1-females with males from the parental populations produced F2-females, and hybrid males withn=23, 24, or 25 chromosomes. The chromosome polymorphism is not due to B-chromosomes. Probably then=25 karyotype originated from then=23 karyotype by two Robertsonian fissions (2 ¯M 4 ¯A), since then=25 karyotype was found in only one of the populations. Diploid males occurred frequently in colonies from four out of five sites investigated.     

Novel oligonucleotide probes for in situ detection of pederin-producing endosymbionts of Paederus riparius rove beetles (Coleoptera: Staphylinidae)

Bacterial endosymbionts from female Paederus rove beetles are hitherto uncultured, phylogenetically related to Pseudomonas sp., and produce the polyketide pederin, which exhibits strong cytotoxic effects and antitumoral activities. The location of such endosymbionts inside beetles and on beetles' eggs is hypothesized based on indirect evidence rather than elucidated. Thus, an endosymbiont-specific and a competitor oligonucleotide probe (Cy3-labelled PAE444 and unlabelled cPAE444, respectively) were designed and utilized for FISH with semi-thin sections of Paederus riparius eggs. Cy3-PAE444-positive cells were densely packed and covered the whole eggshell. Hundred percent of EUB338-Mix-positive total bacterial cells were PAE444 positive, indicating a biofilm dominated by Paederus endosymbionts. Analysis of different egg deposition stadiums by electron microscopy and pks (polyketide synthase gene, a structural gene associated with pederin biosynthesis)-PCR supported results obtained by FISH and revealed that the endosymbiont-containing layer is applied to the eggshell inside the efferent duct. These findings suggest that P. riparius endosymbionts are located inside unknown structures of the female genitalia, which allow for a well-regulated release of endosymbionts during oviposition. The novel oligonucleotide probes developed in this study will facilitate (1) the identification of symbiont-containing structures within genitalia of their beetle hosts and (2) directed cultivation approaches in the future.     

Targeting modular polyketide synthases with iteratively acting acyltransferases from metagenomes of uncultured bacterial consortia

Bacterial type I polyketide synthases (PKSs) produce a wide range of biomedically important secondary metabolites. These enzymes possess a modular structure that can be genetically re-engineered to yield novel drug candidates not found in nature. Recently, we have reported the putative pederin PKS from an uncultured bacterial symbiont of Paederus fuscipes beetles. It belongs to an architecturally unusual PKS group, the members of which contain iteratively acting acyltransferases that are not integrated into the PKS modules but are encoded by isolated genes. As these systems are rare, often contain additional unusual features and are of smaller size than regular PKSs, the development of a method for the targeted isolation of new group members would be of great interest. Here, we present a phylogenetic approach to identify these systems rapidly in highly complex metagenomic DNA samples. To demonstrate its practical value, we located two pederin-type PKS systems putatively involved in the biosynthesis of antitumour polyketides in the metagenomic DNA of beetles, sponges and their uncultivated bacterial symbionts.     

Performance of <Emphasis Type="Italic">Eriopis connexa</Emphasis> (Coleoptera: Coccinellidae) resistant to lambda-cyhalothrin after extended recovery from knockdown

A population of the predatory lady beetle Eriopis connexa (Germar) (Coleoptera: Coccinellidae) was recorded as resistant to lambda-cyhalothrin. Adults exposed to this insecticide have recovered from knockdown after 72 h. Thus, the performance of resistant (R) and susceptible (S) populations of E. connexa not exposed to insecticide (R0 and S0) and R adults recovering from knockdown 24, 48, and 72 h after exposure (R24, R48, and R72) was studied. In addition, the fertility life table parameters were calculated for one generation considering the progenies from R0, S0, and R24 populations. The recovery rate from knockdown was 69.4% for R-adults, and greater recovery rate was observed within 48 h following lambda-cyhalothrin exposure. The S-females produced about 50% more eggs and lived longer, when compared with R-females irrespective of the recovery periods after knockdown. The R-females produced similar number of eggs and exhibited similar longevity across all treatments (R0, R24, R48, and R72). Progenies produced by R- and S-populations did not exhibit consistent differences in development and survival. The fertility life table parameters showed higher intrinsic rate of population growth (r_m) and lower mean generation time (T) for R0- and R24-females, when compared with those for S0-females. Thus, the time interval needed to recover from knockdown is not related to the adaptive cost of resistance in E. connexa.     

Modulation of Fas-dependent apoptosis: a dynamic process controlling both the persistence and death of CD4 regulatory T cells and effector T cells

We have previously shown that regulatory CD25(+)CD4(+) T cells are resistant to clonal deletion induced by viral superantigen in vivo. In this work we report that isolated CD25(+)CD4(+) T cells activated in vitro by anti-CD3 Ab are resistant to Fas-induced apoptosis, in contrast to their CD25(-)CD4(+) counterparts. Resistance of CD25(+)CD4(+) T cells to Fas-dependent activation-induced cell death is not linked to their inability to produce IL-2 or to their ability to produce IL-10. The sensitivity of both populations to Fas-induced apoptosis can be modulated in vitro by changing the CD25(+)CD4(+):CD25(-)CD4(+) T cell ratio. The sensitivity of CD25(-)CD4(+) T cells to apoptosis can be reduced, while the sensitivity of CD25(+)CD4(+) T cells can be enhanced. Modulation of Fas-dependent apoptosis is associated with changes in cytokine production. However, while CD25(-)CD4(+) T cell apoptosis is highly dependent on IL-2 (production of which is inhibited by CD25(+)CD4(+) T cells in coculture), modulation of CD25(+)CD4(+) T cell apoptosis is IL-2 independent. Taken together, these results suggest that CD25(+)CD4(+) and CD25(-)CD4(+) T cell sensitivity to Fas-dependent apoptosis is dynamically modulated during immune responses; this modulation appears to help maintain a permanent population of regulatory T cells required to control effector T cells.     

Induced and spontaneous movements of potassium ions into mitochondria

1. Net movements of K(+) into metabolizing liver mitochondria before and after the addition of valinomycin have been measured by using selective glass electrodes. The advantage of using an automatic titrator to hold the K(+) concentration constant is demonstrated. 2. According to the energy source provided the induced movement after the addition of valinomycin can be either in or out. 3. Uptakes and rates of movement are higher in media containing acetate (20mm) than in media containing chloride (20mm). In each mixture comparisons were made at three pH values; at pH6.36 the induced rates are less than at pH7.0 or 7.8 but the final uptakes attained are increased. 4. The rate of uptake is increased by inorganic phosphate. 5. The presence of Mg(2+) slightly decreases the induced uptake and rate of movement; Ca(2+) can cause the induced movement of K(+) to be outward. 6. The rate of induced K(+) movement is related to the rate of extra oxygen consumption but with different factors in acetate (24 K(+) ions/oxygen molecule) and chloride media (10 K(+) ions/oxygen molecule). 7. The amount of K(+) gained is proportional to the loss of fluorescence of the suspension. 8. When K(+) moves there is a contrary movement of H(+) but the ratio depends on the conditions. At pH6.36 in chloride media the K(+)/H(+) ratio exceeded 10:1 and in no case did it fall to unity. 9. When K(+) is taken up there is a proportional diminution of light-scattering; it is inferred that swelling takes place along with K(+) accumulation. 10. It is shown by the use of tracer (42)K(+) that turnover of the ion in mitochondria is increased by valinomycin. 11. It is concluded that valinomycin both increases the permeability to K(+) and also, given an adequate energy supply, stimulates the K(+)-accumulating mechanism.     

Mannose receptor expression and function define a new population of murine dendritic cells

In vitro the mannose receptor (MR) mediates Ag internalization by dendritic cells (DC) and favors the presentation of mannosylated ligands to T cells. However, in vivo MR seems to play a role not in Ag presentation but in the homeostatic clearance of endogenous ligands, which could have the secondary benefit of reducing the levels of endogenous Ag available for presentation to the adaptive immune system. We have now observed that while MR(+) cells are consistently absent from T cell areas of spleen and mesenteric lymph nodes (LN), peripheral LN of untreated adult mice contain a minor population of MR(+)MHCII(+) in the paracortex. This novel MR(+) cell population can be readily identified by flow cytometry and express markers characteristic of DC. Furthermore, these MR(+) DC-like cells located in T cell areas can be targeted with MR ligands (anti-MR mAb). Numbers of MR(+)MHCII(+) cells in the paracortex are increased upon stimulation of the innate immune system and, accordingly, the amount of anti-MR mAb reaching MR(+)MHCII(+) cells in T cell areas is dramatically enhanced under these conditions. Our results indicate that the MR can act as an Ag-acquisition system in a DC subpopulation restricted to lymphoid organs draining the periphery. Moreover, the effect of TLR agonists on the numbers of these MR(+) DC suggests that the immunogenicity of MR ligands could be under the control of innate stimulation. In accordance with these observations, ligands highly specific for the MR elicit enhanced humoral responses in vivo only when administered in combination with endotoxin.     

The effect of changes of environment on the electrical and ionic pattern of muscle

The resting and action potentials of sartorius muscles of the toad, Bufo marinus, have been measured under varying conditions of external environment. At the same time, analyses for Na(+) and K(+) content were carried out. There was a slight elevation of 2 mv. when the measurements were made in phosphate-Ringer instead of in bicarbonate-Ringer. The R.P. was independent of the hydrogen ion concentration between pH 6.5 and 8.5, although at these pH's there was marked alteration in the level of Na(+) and K(+) in the muscle. Alteration of the external K(+) level between 0 and 50 m.eq./liter has little influence on the internal K(+) concentration. When the log of the external K(+) concentration is plotted against the R.P. there is not a linear relationship until the external K(+) is raised above 12 m.eq./liter, at which point the cell is unexcitable. Above this value a straight line with a slope of 58 mv. per ten-fold change in concentration is obtained, but the absolute values at any point are about 35 per cent higher than those which would be given by the Nernst equation. Alteration of the external Na(+) level within a range of 45 to 650 m.eq./liter resulted in marked changes in the internal Na(+) content, without, however, having any effect on the ratio Na(+) (out)/Na(+) (in). This ratio has remained at about 3 in spite of marked fluctuations in the absolute value of the internal and external Na(+) levels. When the Na(+) level is lowered there is a decrease in the height of the action potential although there is no alteration in the ratio Na(+) (out)/Na(+) (in). As the Na(+) level is raised the height of the action potential is not affected even in the presence of a fivefold increase in Na(+) in the Ringer. The results do not support the conclusion that the bioelectric potentials can be calculated from the ionic ratios by means of simple physical chemical hypotheses such as the Nernst or Goldman equations. The maintenance of the normal K(+) content of the cell cannot be accounted for by a Donnan mechanism. No definite evidence has been produced to explain the mechanism of a Na(+) "pump." In other words, the concept of a Na(+) pump requires that there shall be a physico- or organochemical mechanism which will distinguish between Na(+) and K(+) (or other) ions. There is evidence that Na(+) can be extruded against a concentration gradient. On the other hand the cell is able to maintain a constant ratio of external to internal Na(+) even when the cell has been severely damaged by very high external Na(+) levels.     

Identification of Mycobacterium tuberculosis-specific Th1, Th17 and Th22 cells using the expression of CD40L in tuberculous pleurisy

Important advances have been made in the immunodiagnosis of tuberculosis (TB) based on the detection of Mycobacterium tuberculosis (MTB)-specific T cells. However, the sensitivity and specificity of the immunological approach are relatively low because there are no specific markers for antigen-specific Th cells, and some of the Th cells that do not produce cytokines can be overlooked using this approach. In this study, we found that MTB-specific peptides of ESAT-6/CFP-10 can stimulate the expression of CD40L specifically in CD4(+) T cells but not other cells from pleural fluid cells (PFCs) in patients with tuberculous pleurisy (TBP). CD4(+)CD40L(+) but not CD4(+)CD40L(-) T cells express IFN-γ, IL-2, TNF-α, IL-17 or IL-22 after stimulation with MTB-specific peptides. In addition, CD4(+)CD40L(+) T cells were found to be mostly polyfunctional T cells that simultaneously produce IFN-γ, IL-2 and TNF-α and display an effector or effector memory phenotype (CD45RA(-)CD45RO(+)CCR7(-)CD62L(-)ICOS(-)). To determine the specificity of CD4(+)CD40L(+) T cells, we incubated PFCs with ESTA-6/CFP-10 peptides and sorted live CD4(+)CD40L(+) and CD4(+)CD40L(-) T cells by flow cytometry. We further demonstrated that sorted CD4(+)CD40L(+), but not CD4(+)CD40L(-) fractions, principally produced IFN-γ, IL-2, TNF-α, IL-17 and IL-22 following restimulation with ESTA-6/CFP-10 peptides. Taken together, our data indicate that the expression of CD40L on MTB-specific CD4(+) T cells could be a good marker for the evaluation and isolation of MTB-specific Th cells and might also be useful in the diagnosis of TB.     

Sodium-potassium synergism in Theobroma cacao: stimulation of photosynthesis, water-use efficiency and mineral nutrition

In ecological setting, sodium (Na(+) ) can be beneficial or toxic, depending on plant species and the Na(+) level in the soil. While its effects are more frequently studied at high saline levels, Na(+) has also been shown to be of potential benefit to some species at lower levels of supply, especially in C4 species. Here, clonal plants of the major tropical C3 crop Theobroma cacao (cacao) were grown in soil where potassium (K(+) ) was partially replaced (at six levels, up to 50% replacement) by Na(+) , at two concentrations (2.5 and 4.0 mmol(c) dm(-3) ). At both concentrations, net photosynthesis per unit leaf area (A) increased more than twofold with increasing substitution of K(+) by Na(+) . Concomitantly, instantaneous (A/E) and intrinsic (A/g(s) ) water-use efficiency (WUE) more than doubled. Stomatal conductance (g(s) ) and transpiration rate (E) exhibited a decline at 2.5 mmol dm(-3) , but remained unchanged at 4 mmol dm(-3) . Leaf nitrogen content was not impacted by Na(+) supplementation, whereas sulfur (S), calcium (Ca(2+) ), magnesium (Mg(2+) ) and zinc (Zn(2+) ) contents were maximized at 2.5 mmol dm(-3) and intermediate (30-40%) replacement levels. Leaf K(+) did not decline significantly. In contrast, leaf Na(+) content increased steadily. The resultant elevated Na(+) /K(+) ratios in tissue correlated with increased, not decreased, plant performance. The results show that Na(+) can partially replace K(+) in the nutrition of clonal cacao, with significant beneficial effects on photosynthesis, WUE and mineral nutrition in this major perennial C3 crop.     

Activation of CD25(+)CD4(+) regulatory T cells by oral antigen administration

CD25(+)CD4(+) T cells are naturally occurring regulatory T cells that are anergic and have suppressive properties. Although they can be isolated from the spleens of normal mice, there are limited studies on how they can be activated or expanded in vivo. We found that oral administration of OVA to OVA TCR transgenic mice resulted in a modification of the ratio of CD25(+)CD4(+) to CD25(-)CD4(+) cells with an increase of CD25(+)CD4(+) T cells accompanied by a decrease of CD25(-)CD4(+) T cells. The relative increase in CD25(+)CD4(+) T cells persisted for as long as 4 wk post feeding. We also found that CTLA-4 was dominantly expressed in CD25(+)CD4(+) T cells and there was an increase in the percentage of CD25(+)CD4(+) T cells expressing CTLA-4 in OVA-fed mice. In contrast to CD25(-)CD4(+) cells, CD25(+)CD4(+) cells from fed mice proliferated only minimally to OVA or anti-CD3 and secreted IL-10 and elevated levels of TGF-beta(1) following anti-CD3 stimulation. CD25(+)CD4(+) cells from fed mice suppressed the proliferation of CD25(-)CD4(+) T cells in vitro more potently than CD25(+)CD4(+) T cells isolated from unfed mice, and this suppression was partially reversible by IL-10 soluble receptor or TGF-beta soluble receptor and high concentration of anti-CTLA-4. With anti-CD3 stimulation, CD25(+)CD4(+) cells from unfed mice secreted IFN-gamma, whereas CD25(+)CD4(+) cells from fed mice did not. Adoptive transfer of CD25(+)CD4(+) T cells from fed mice suppressed in vivo delayed-type hypersensitivity responses in BALB/c mice. These results demonstrate an Ag-specific in vivo method to activate CD25(+)CD4(+) regulatory T cells and suggest that they may be involved in oral tolerance.     

Differentiation of monocytic cell clones into CD8 alpha+ dendritic cells (DC) suggests that monocytes can be direct precursors for both CD8 alpha+ and CD8 alpha- DC in the mouse

Dendritic cells (DC) are the professional APCs that initiate T cell immune responses. DC can develop from both myeloid and lymphoid progenitors. In the mouse, the CD8alpha(+) DC had been designated as "lymphoid" DC, and CD8alpha(-) DC as "myeloid" DC until recently when it was demonstrated that common myeloid progenitors can also give rise to CD8alpha(+) DC in bone marrow chimera mice. However, it is still not clear which committed myeloid lineages differentiate into CD8alpha(+) DC. Because monocytes can differentiate into DC in vivo, the simplest hypothesis is that the CD8alpha(+) DC can be derived from the monocyte/macrophage. In this study we show that cell clones, isolated from CD8alpha(+) DC lymphoma but with a monocytic phenotype (CD11c(low/-)D11b(high)CD8alpha(-)I-A(low)), can redifferentiate into CD8alpha(+) DC either when stimulated by LPS and CD40L or when they migrate into the lymphoid organs. Maturation of DC in vivo correlated with strong priming of allogeneic T cells. Moreover, the monocytes from cultured splenocytes or peritoneal exudates macrophages of wild-type mice are also capable of differentiating into CD11c(+)CD8alpha(+) DC after their migration into the draining lymph nodes. Our results suggest that monocytes can be direct precursors for CD11c(+)CD8alpha(+) DC in vivo. In addition, the monocyte clones described in this study may be valuable for studying the differentiation and function of CD8alpha(+) DC that mediate cross-presentation of Ag to CD8 T cells specific for cell-associate Ags.     

Extracellular pH modulates kinetics of the Na(+),K(+)-ATPase

To investigate effects of pH on the Na(+),K(+)-ATPase, we used the Xenopus oocytes to measure transient charge movements in the absence of extracellular K(+), and steady-state currents mediated by the pump as well as ATPase activity. The activity of purified Na(+), K(+)-ATPase strongly depends on pH, which has been attributed to protonation of intracellular sites. The steady-state current reflects pump activity, the transient charge movement voltage-dependent interaction of external Na(+) ions with the pump molecule and/or conformational changes during Na(+)/Na(+) exchange. The steady-state current exhibits a characteristic voltage dependence with maximum at about 0 mV at low external K(+) (< or =2 mM) and with 50 Na(+). This dependency is not significantly affected by changes in external pH in the range from pH 9 to pH 6. Only below pH 6, the voltage dependence of pump current becomes less steep, and may be attributed to a pH-dependent inhibition of the forward pump cycle by external Na(+). External stimulation of the pump by K(+) in the absence of Na(+) can be described by a voltage-dependent K(m) value with an apparent valency z(K). At higher external pH the z(K) value is reduced. The transient current signal in the absence of external K(+) can be described by the sum of three exponentials with voltage-dependent time constants of about 50 ms, 700 micros and less than 100 micros during pulses to 0 mV. The charge distribution was calculated by integration of the transient current signals. The slowest component and the associated charge distributions do not significantly depend on external pH changes. The intermediate component of the transients is represented by a voltage-dependent rate constant which shows a minimum at about -120 mV and increases with decreasing pH. Nevertheless, the contribution to the charge movement is not altered by pH changes due to a simultaneous increase of the amplitude of this component. We conclude that reduction of external pH counteracts external K(+) and Na(+) binding.