Changes In External pH Research Articles

Effects of intracellular pH on ATP‐sensitive K+ channels in mouse pancreatic beta‐cells.

1. The effects of intracellular pH (pHi) on the ATP-sensitive K+ channel (K+ATP channel) from mouse pancreatic beta-cells were examined in inside-out patches exposed to symmetrical 140 mM K+ solutions. 2. The relationship between channel activity and pHi was described by the Hill equation with half-maximal inhibition (Ki) at pHi 6.25 and a Hill coefficient of 3.7. 3. Following exposure to pHi < 6.8, channel activity did not recover to its original level. Subsequent application of trypsin to the intracellular membrane surface restored channel activity to its initial level or above. 4. At -60 mV the relationship between pHi and the single-channel current amplitude was described by a modified Hill equation with a Hill coefficient of 2.1, half-maximal inhibition at pHi 6.48 and a maximum inhibition of 18.5%. 5. A decrease in pHi reduced the extent of channel inhibition by ATP: Ki was 18 microM at pH 7.2 and 33 microM at pH 6.4. The Hill coefficient was also reduced, being 1.65 at pH 7.2 and 1.17 at pH 6.4. 6. When channel activity was plotted as a function of ATP4- (rather than total ATP) there was no effect of pHi on the relationship. This suggests that ATP4- is the inhibitory ion species and that the effects of reducing pHi are due to the lowered concentration of ATP4-. 7. Changes in external pH had little effect on either single-channel or whole-cell K+ATP currents. 8. The effects of pHi do not support a role for H+ in linking glucose metabolism to K+ATP channel inhibition in pancreatic beta-cells.

Control of lactate utilization by extracellular pH in isolated rat liver cells

This study reports the influence of external pH on lactate balance in hepatocytes isolated from fed and 24-hour-starved rats. The effects of changes in extracellular pH on the utilization of lactate by liver cells has been studied in conditions stimulating metabolic acidosis (pH 7.15, 10 mmol/L bicarbonate). The addition of lactate to a suspension of liver cells from fed rats shifted the lactate balance from net release to net utilization; the threshold of this shift was about 3 mmol/L in the presence of 10 mmol/L glucose. In these cells, acidic external pH (7.15) played a crucial role in stimulating the lactate utilization as shown by (1) a diminished release of lactate in the absence of lactate addition (−60%); (2) a marked decrease of the threshold of lactate utilization down to 1.2 mmol/L; and (3) a net stimulation of the lactate utilization for concentrations in the physiologic range (2 to 3 mmol/L). The effect of acidosis was mediated by an inhibition of glycolysis (−40%). Besides that, at pH 7.45, the addition of 100 μmol/L AICA-riboside 5-amino-4-imidazolecarboxamide riboside, (an inhibitor of hepatic glycolysis) mimicked the effect of acidosis. Moreover, differences in lactate fluxes between the two pH conditions were decreased in the absence of glucose. In liver cells from starved rats, regardless of the concentration of added lactate, the lactate balance was always directed toward net utilization. Accordingly, a change in external pH from 7.45 to 7.15 had a lesser effect on lactate metabolism than in liver cells from fed rats. However, a significant increase of lactate utilization was observed in hepatocytes from starved rats when lactate concentrations were up to 3 mmol/L. This effect could also be related to the decrease of hepatic glycolysis. In conclusion, this study indicates a crucial role of a low extracellular pH in controlling the hepatic metabolism toward lactate utilization in hepatocytes isolated from fed rats.

Systems analysis of yeast gylcolysis using perturbations of external pH: physiological parameters, non-parametric analysis, and general properties of the system dynamics

A global systems analysis approach to yeast glycolysis is presented. Starting from a high flux steady state of anaerobic glycolysis rapid changes of external pH are applied. The dynamics of the transduction of the pH signal to the in vivo fluorescence of reduced pyridine nucleotides are investigated. Physiological studies indicate no significant influence of external pH on glycolytic flux and energy charge, but stationary concentrations of metabolites are increased after increasing the external pH from 5–7 particularly in the distal part of the glycolytic chain. Most of the changes in reduced pyridine fluorescence are due to glycolytically accessible NADH. As a first stage of system analytic investigations the premises for the approach were tested. The dynamic properties of the system are stable and the response is linear with respect to the input amplitude. The dynamic properties of the system depend on the state of adaptation to a certain external pH. The non-parametric model derived from single step experiments was used to correctly predict the response to pulses of variable duration. This indicates that the linear macroscopic approach to systems analysis with pH as input is applicable. The dynamics of the system response includes strongly damped oscillatory components. Their period lengths are similar to those found with temperature inputs and with the switching from low to high glycolytic flux under anaerobic conditions. This indicates that the three different signals may trigger common regulatory interactions. The order of the signal transduction process, indicating the number of basic processes involved, can be estimated to be 4 or higher.

The influence of DNA topology on the environmental regulation of a pH-regulated locus in Salmonella typhimurium.

Salmonella typhimurium is exposed to major shifts in H+ concentration both in its natural and pathogenic environments. The organism undergoes extensive changes in gene expression in response to these pH fluctuations. A current question of regulatory biology is how a change in external pH selectively modulates transcription. We have analysed the expression of one such pH-regulated locus, aniG, and found it is controlled by several additional environmental conditions including osmolarity and oxygen. For factors such as osmolarity and anaerobiosis, an environmentally triggered change in DNA supercoiling has been suggested as a means for controlling gene expression. Thus, environmentally induced changes in DNA topology were explored as a possible common means for establishing the multiple controls on aniG. The involvement of DNA supercoiling in the genetic response of S. typhimurium to external pH has not previously been defined. This report establishes that alkaline environments lower the linking number of reporter plasmids when compared to acidic environments. A consistent pattern was then established whereby conditions or mutations leading to either increased or decreased negative supercoiling were associated with altered expression of aniG. A similar relationship was observed for another environmentally regulated locus, proU. The DNA topology effects on aniG expression were dependent on the presence of EarA, the negative regulator of aniG. These data can be explained by a model in which repressor-operator interactions are very sensitive to changes in operator conformation. These environmentally induced topological influences on operator DNA structure contribute to the magnitude of pH control exerted upon aniG.

Calcium–hydrogen exchange by the plasma membrane Ca-ATPase of voltage-clamped snail neurons

The submicromolar levels of free Ca(2+) ions in animal cells are believed to be maintained in the long term by two different plasma membrane transport mechanisms. These are Na-Ca exchange, driven by the sodium gradient, and a Na-independent Ca pump, driven by ATP. There is good evidence from red blood cells, and indirect evidence from other non-neuronal preparations, that the Ca-ATPase exchanges internal Ca(2+) for external H(+). Although Ca extrusion from nerve cells is inhibited by high external pH, there as yet is no evidence for the counter-transport of H(+). We have used both pH- and calcium-sensitive microelectrodes on the cell surface, and the Ca indicator fura-2 intracellularily, to investigate how snail neurons regulate cytoplasmic free Ca(2+). We now report that in snail neurons the recovery of intracellular Ca(2+) after an increase coincides with both the expected increase in surface Ca(2+) and a decrease in surface H+. Recovery of intracellular Ca and the changes in surface pH and Ca are all blocked by intracellular vanadate. We conclude that snail neurons regulate intracellular Ca mainly by a Ca-H ATPase, and suggest that this Ca-H exchange may account for many of the reported extracellular pH changes seen with neuronal excitation.

Regulation of the electrogenic H+ channel in the plasma membrane of neutrophils: possible role of phospholipase A2, internal and external protons

Possible factors regulating the opening of and the rate of H+ flux through a recently described, Cd(2+)-sensitive, phorbol ester- and arachidonic acid (AA)-activatable H(+)-conducting pathway in the plasma membrane of neutrophil granulocytes were investigated. (1) The phospholipase A2 blocker p-bromophenacyl bromide (BPB) inhibited the phorbol 12-myristate 13-acetate (PMA)-induced activation of this channel in a concentration-dependent manner (IC50, 4 microM). (2) Neither BPB nor the protein kinase C (PKC) inhibitor staurosporine influenced the AA-elicited stimulation of this route. (3) Intracellular acidification (cytoplasmic pH below 6.9) itself is capable of activating an electrogenic, Cd(2+)-sensitive H+ efflux indicating that protons can open up this route in the absence of any other stimulator. (4) PMA significantly decreases the intracellular H+ concentration ([H+]i) threshold for the opening of the channel, thus providing a conductive state at resting pH values, and elevates the rate of H+ efflux at any [H+]i. (5) Changes in external pH also modify the operation of the channel: above an extracellular pH (pH(o)) value of 7.4, the H(+)-flux/driving force relationship is approx. 5-fold greater than below this value. Our results suggest a multifactorial regulation of the electrogenic H+ channel: most probably PKC activates the channel indirectly, via stimulation of phospholipase A2 that subsequently liberates AA. In addition to this, the channel conductance seems to be promoted by internal H+ and inhibited by external H+.

Influence of pH on bacterial gene expression

Bacteria respond to changes in internal and external pH by adjusting the activity and synthesis of proteins associated with many different processes, including proton translocation, amino acid degradation, adaptation to acidic or basic conditions and virulence. While, for many of these examples, the physiological and biological consequence of the pH-induced response is clear, the mechanism by which the transcription/translation machinery is signalled is not. These examples are discussed along with several others in which the function of the gene or protein remains a mystery.

Evidence for a K(+)-H+ exchange in trout red blood cells.

1. Exposure of trout red blood cells to beta-adrenergic agonist isoprenaline activates a cAMP-dependent Na(+)-H+ antiport, the movements of protons being compensated by a Cl(-)-OH- (or HCO3-) exchange mediated by band 3 protein. The absorption of water osmotically linked to sodium and chloride induces cell swelling. 2. In the presence of acetazolamide, anionic exchange is inhibited and activation of cationic exchange resulted in the first 2 min in a strong external acidification and a large internal alkalinization leading to a reversal of the transmembrane pH gradient. Then, for at least 1 h and despite the inhibition of Cl- entry, a net Na+ uptake occurred which was balanced by an equivalent K+ loss, with the result that cell volume and pH gradient remained unchanged. 3. In such conditions, the inactivation of the Na(+)-H+ exchanger by a beta-antagonist, propranolol, blocked Na+ entry while K+ continued to be lost. This volume-independent K+ efflux, which is thus independent of the Na(+)-H+ exchanger, was not accompanied by a Cl- efflux but was associated with large internal and external pH changes consistent with K(+)-H+ exchange. 4. The K+ loss and the related pH changes are inhibited by compounds which are known to inhibit the K(+)-anion co-transporter in trout red cells, i.e. 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (Dids) and niflumic acid.

Regulation of Calcium Influx in Chara

Measurements were made of (45)Ca influx into isolated internodal cells of Chara corallina and also into internodal cells of intact plants. (45)Ca influx was closely related to growth. In rapidly expanding internodal cells, the influx was approximately 1.4 nmol m(-2) s(-1) compared to the influx in mature cells from slow-growing cultures of 0.2 nmol m(-2) s(-1). Isolated internodal cells had influxes in the range 0.2 to 0.7 nmol m(-2) s(-1), but this increased to approximately 2 nmol m(-2) s(-1) in high calcium solutions and to 4 nmol m(-2) s(-1) in high potassium solutions. No significant effects on calcium influx were observed for changes in external pH or for treatments that changed internal pH, except that NH(4) was slightly inhibitory. Severe metabolic inhibition by carbonylcyanide-m-chlorophenyl-hydrazone stimulated influx, whereas dicyclohexylcarbodiimide had no effect and darkness inhibited influx. La(3+) also inhibited influx, but the organic channel blockers nifedipine and bepridil stimulated influx. Verapamil had no effect. The results are generally consistent with voltage regulation of calcium channels as in animal cells.

Protein kinase inhibitor K‐252a and fusicoccin induce similar initial changes in ion transport of parsley suspension cells

The protein kinase inhibitor K‐252a induces a rapid, transient decrease of extracellular pH and [K+], and a concomitant increase in extracellular [Ca2+] in suspensions of cultured parsley cells. These effects are subsequently reversed. As with K‐252a, fusicoccin also induces similar changes in pH and extracellular [Ca2+], but reversion does not occur. Acidification by HCI also leads to an increase in external [Ca2+], suggesting that the changes in extracellular [Ca2+] are mainly due to a pH‐dependent displacement of Ca2+ ionically bound to the cell wall. The artificial acidification by HCI is rapidly followed by cell‐mediated alkalinization, a process associated with K2 release and rebinding of Ca2+. Any change in external pH or [K+] induced by K‐252a, fusicoccin, or HCI is followed by an uptake of 45Ca2+ into cellular pools. The results show that K‐252a may be a valuable tool for studying the complex regulation of ion transport which may involve changes in the phosphorylation of unknown proteins.

Depolarization-induced calcium release from isolated triads measured with impermeant Fura-2

Depolarization-induced Ca2+ release was studied in a mixture of triads and terminal cisternae isolated from rabbit skeletal muscle. The vesicles were actively loaded with known amounts of Ca2+ in the absence of precipitating anions in a solution containing 100 mM K propionate buffer. Changes in extravesicular Ca2+ were monitored with 10 microM Fura-2 (membrane impermeant form). Ca2+ release was initiated by diluting an aliquot of the loaded vesicles into a TEACl release solution designed to maintain a constant [K+].[Cl-] product. Fast release, defined as the percentage of total Ca2+ loaded which released in less than 10 sec, occurred when extravesicular free Ca2+ was in the submicromolar range and was unaffected by 5 mM caffeine under depolarizing conditions, change in external pH to 6.5, and an increase in external Mg2+ concentration from 0.1 to 0.2 mM. Thus, the Ca2+ release measured in these studies is distinct from Ca(2+)-induced Ca2+ release. The fast release more than doubled when a greater dilution (1:20 versus 1:10) of the loaded vesicles into the release solution, which would produce a larger depolarization, was used. The percentage of loaded Ca2+ which released rapidly in a particular triad preparation was similar to the percentage of vesicles structurally coupled as visualized by electron microscopy.

Influence of extracellular and intracellular pH on GABA-gated chloride conductance in crayfish muscle fibres

The effect of intracellular and extracellular pH on GABA-gated Cl − conductance was studied using H +-selective microelectrodes and a three-microelectrode voltage clamp in crayfish leg opener muscle fibres in bicarbonate-free solutions. Experimental variation of intracellular pH in the range 6.4–8.0 did not affect the GABA-gated conductance. In contrast to this, the GABA-gated conductance was sensitive to changes in external pH. Raising the external pH from 7.4 to 8.4 decreased the GABA-gated peak conductance observed immediately following application of GABA by 30%, and a change from 7.4 to 6.4 produced an increase of 26%. The effect of extracellular pH on the GABA-gated peak conductance was approximately linear in the pH range 6.4–8.9. A slight decrease in the slope of the pH-conductance relationship was evident in the pH range 5.4–6.4. The desensitization of the GABA-gated conductance was also affected by external pH. At pH 6.9 the conductance produced by 1 mM GABA showed a desensitization of about 15%, and at pH 8.9 this value was 34%. Raising the external pH in the presence of GABA decreased the GABA-gated peak conductance and increased the fractional desensitization, while lowering the external pH produced opposite effects, and was capable of repriming the conductance from a desensitized state to the non-desensitized state. The above results show that the GABA-gated conductance is sensitive to changes in external pH in the physiological range, and suggest that pH-dependent changes in the postsynaptic efficacy of GABA-mediated inhibition may contribute to H + modulation of neuronal excitability.

Bicarbonate-dependent changes of intracellular sodium and pH in identified leech glial cells.

A new triple-barrelled ion-sensitive microelectrode was used to investigate the importance of bicarbonate for the regulation of intracellular Na+ and pH (Nai and pHi, respectively) of neuropile glial cells in the central nervous system of the leech Hirudo medicinalis. Addition of CO2/HCO3- produced an increase of the Nai activity and an intracellular alkalinization, indicating bicarbonate accumulation in the glial cells. Changes of external pH (from 7.4 to 7.0 and 7.8) produced large and rapid shifts of pHi and Nai and of the membrane potential in the presence, but not in the absence, of bicarbonate. Thus, acid/base transport and Na+ movements across the glial membrane into and out of the cells were accelerated severalfold in CO2/HCO3(-)-buffered saline as compared to a CO2/HCO3(-)-free, HEPES-buffered saline. The results suggest that the electrogenic, reversible, cotransport of Na+ and HCO3- in the glial cell membrane [3,9] can produce significant changes in intraglial pH and Na activity, and can carry a significant fraction of the total Na+ flux across the cell membrane.

フラグミン静注の配合試験 （第１報） 各種輸液およびビタミン剤との配合変

Fragmin IV, dalteparin sodium for injection (1000 international units/ml), was studied on its compatibility with various transfusions and vitamin injections.Every combination showed neither change in external appearance and pH nor significant decrease in residual anti-factor Xa activity up to 24 hours after mixing.Fragmin IV, so compatible with various transfusions and vitamin injections, is believed to be practically useful for treatment of disseminated intravascular coagulation (DIC).

Electron and proton transport across the plasma membrane

Transplasm membrane electron transport in both plant and animal cells activates proton release. The nature and components of the electron transport system and the mechanism by which proton release is activated remains to be discovered. Reduced pyridine nucleotides are substrates for the plasma membrane dehydrogenases. Both plant and animal membranes have unusual cyanide-insensitive oxidases so oxygen can be the natural electron acceptor. Natural ferric chelates or ferric transferrin can also act as electron acceptors. Artificial, impermeable oxidants such as ferricyanide are used to probe the activity. Since plasma membranes contain b cytochromes, flavin, iron, and quinones, components for electron transport are present but their participation, except for quinone, has not been demonstrated. Stimulation of electron transport with impermeable oxidants and hormones activates proton release from cells. In plants the electron transport and proton release is stimulated by red or blue light. Inhibitors of electron transport, such as certain antitumor drugs, inhibit proton release. With animal cells the high ratio of protons released to electrons transferred, stimulation of proton release by sodium ions, and inhibition by amilorides indicates that electron transport activates the Na+/H+ antiport. In plants part of the proton release can be achieved by activation of the H+ ATPase. A contribution to proton transfer by protonated electron carriers in the membrane has not been eliminated. In some cells transmembrane electron transport has been shown to cause cytoplasmic pH changes or to stimulate protein kinases which may be the basis for activation of proton channels in the membrane. The redox-induced proton release causes internal and external pH changes which can be related to stimulation of animal and plant cell growth by external, impermeable oxidants or by oxygen.

特集： 膜機能の制御 微生物の環境適応と膜機能

Microorganisms have potential capacities to adapt to environmental changes to a certain extent. As extreme cases, a variety of microorganisms such as thermophiles, psychrophiles, acidophiles, alkalophiles and halophiles have been isolated from the natural environments. The structure and function of cell membranes play essential roles to cope with an environmental stress. We studied the modes of adaptations of a slightly halophilic marine bacterium, Vibrio alginolyticus, to salt concentrations and pH changes. As a response to osmotic stress, the cells accumulate K+, glutamate and proline as osmoprotectants. Except for extreme halophiles (archaebacteria), the mode of osmotic adaptation of halophilic bacteria was very similar to those of nonhalophilic bacteria. The adaptation to the changes in external pH is performed by maintaining the intracellular pH nearly constant. In V. alginolyticus, K+/H+ antiporter functioned as a “pH regulator” over the pH range from 6.0 to 9.0. Marine bacteria are unique in having the respiratory chain-driven Na+ pump. The site of energy coupling was assigned to the Na+_dependent NADH-quinone reductase segment, which was composed of three subunits (α, β, γ) and contained FAD (β) and FMN (α) as cofactors. Since the respiratory chain of marine bacteria also generates proton-motive force, both Na+ and H+ are utilized as energy-coupling ions. Energy coupling by the Na+ circulation is of great advantage to the energetics of marine bacteria. These examples represent the importance of membrane functions in the microbial adaptations to environmental changes.

Extracellular alkaline-acid pH shifts evoked by iontophoresis of glutamate and aspartate in turtle cerebellum

The effect of glutamate and aspartate iontophoresis on extracellular pH was investigated in the turtle cerebellum in vitro. Both amino acids produced a rapid alkaline transient, typically followed by a prolonged acidification. These responses could be evoked in all layers of the cerebellum. Transition from bicarbonate toN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid-buffered media amplified the pH shifts. Similar alkaline-acid transients could be evoked in the molecular layer by electrical stimulation of the parallel fibers or the ipsilateral peduncle, or by superfusion of glutamate or aspartate. However, no alkaline shifts were evoked in the granular layer by either parallel fiber or peduncle stimulation. In contrast, the iontophoretically induced alkaline shifts were largest in the granular layer. Compared with the stimulus-evoked alkalinizations, the iontophoretic alkaline shifts were relatively insensitive to Mn 2+ or Cd 2+. These data suggest that the activity-dependent alkalinization of brain extracellular space is generated by a bicarbonate-independent mechanism related to excitatory synaptic transmission. The results are consistent with a flux of hydrogen ions through cationic channels, but do not support a direct role for voltage-dependent Ca 2+ channels. In view of the sensitivity of ion channels to changes in external pH, and the magnitude of the amino acid-induced pH shifts, these results indicate that extracellular pH could play an important modulatory role in excitatory synaptic transmission.

Effect of external pH perturbations on in vivo protein synthesis by the acidophilic bacterium Thiobacillus ferrooxidans

The response of the obligate acidophilic bacterium Thiobacillus ferrooxidans to external pH changes is reported. When T. ferrooxidans cells grown at pH 1.5 were shifted to pH 3.5, there were several changes in the general protein synthesis pattern, including a large stimulation of the synthesis of a 36-kDa protein (p36). The apparent low isoelectric point of p36, its location in the membrane fraction, and its cross-reaction with anti-OmpC from Salmonella typhi suggested that it may be a porin whose expression is regulated by extracellular pH.

The effect of external pH changes on responses to excitatory amino acids in mouse hippocampal neurones.

1. The whole-cell and outside-out configurations of the patch-clamp technique were used to record responses to excitatory amino acids in mouse hippocampal neurones in cell culture at different pH. The amino acids kainate, quisqualate, N-methyl-D-aspartate (NMDA) and L-glutamate were applied by a rapid perfusion system. 2. In the whole-cell recording mode the responses to NMDA or to low concentrations of glutamate, recorded in the absence of Mg2+ and with glycine in the extracellular superfusion solution, were antagonized by acidic pH and potentiated by an alkaline extracellular solution. Decrease in pH from 7.3 to 6.0 reduced NMDA responses to 33 +/- 2% and an increase in pH from 7.3 to 8.0 potentiated it to 141 +/- 6%. The responses to quisqualate and kainate were only slightly changed by altering the pH from 7.3 to 6.3 or 8.3. 3. The equilibrium dissociation constant (Kd) for H+ antagonism of responses to NMDA, estimated from the fit of a single-binding-site adsorption isotherm, was calculated to be 0.25 +/- 0.06 microM, corresponding to pH 6.6 +/- 0.1. The H+ attenuation of NMDA current was voltage independent at membrane potentials -60 to +30 mV. 4. H+ antagonism of responses to NMDA was reduced when the NMDA concentration was lowered. In the pH range 6.3-8.3 the H(+)-induced reduction did not vary with the concentration of glycine or Mg2+. The sensitivity of NMDA current to Zn2+ was unchanged in the pH range 6.3 +/- 8.0. These results suggest that H+ ions do not directly interfere with the binding of NMDA to its agonist recognition site or with the binding of glycine, Mg2+ and Zn2+ to the specific allosteric sites on the NMDA receptor-channel complex. 5. In outside-out patches held at -60 mV, unitary NMDA-activated currents were recorded at pH 7.3 and 6.3. The mean NMDA single-channel conductance (gamma) obtained for the largest and most frequent openings were: gamma 7.3 = 52.5 +/- 0.8 pS and gamma 6.3 = 51.8 +/- 0.9 pS. The duration of the mean channel open time, tau o, decreased from 4.75 +/- 0.25 ms in the control at pH 7.3 to 3.59 +/- 0.21 ms at pH 6.3. The mean burst duration, tau b, was reduced from 8.51 +/- 0.78 ms at control pH 7.3 to 5.1 +/- 0.34 ms at pH 6.3. The frequency of NMDA channel bursts was reduced by 31%.(ABSTRACT TRUNCATED AT 400 WORDS)

Modifications of single acetylcholine-activated channels in BC3H-1 cells. Effects of trimethyloxonium and pH.

We have examined the effects of chemical modification with trimethyloxonium (TMO) and changes in external pH on the properties of acetylcholine (ACh)-activated channels in BC3H-1 cells, a clonal muscle cell line. TMO reacts covalently and specifically with carboxylic acid moieties in proteins to convert them to neutral methyl esters. In BC3H-1 cells TMO modification reduces the whole-cell response to ACh measured at negative membrane potentials by approximately 60%. G omega seal patch-clamp recordings of single ACh channel currents showed that the reduction in ACh sensitivity is due to alterations in both the current-carrying and the kinetic properties of the channels. Under all our experimental conditions, i.e., in external solutions of normal or low ionic strength, with or without external divalent cations, and at external pHs between 5.5 and 8.1, TMO treatment reduced ACh single-channel conductance to 70-90% of normal. The effects of TMO on channel kinetics were dependent on the ionic conditions. In normal ionic strength solutions containing both calcium and magnesium ions TMO modification reduced the channel average open time by approximately 25%. A similar reduction in open time was seen in calcium-free solution, but was not present when both calcium and magnesium ions were absent from the external solution. Lowering the ionic strength of the solution increased the mean open time in normal channels by about threefold, but did not affect the kinetics of modified channels. In low ionic strength solutions normal ACh channel open times were maximal at approximately pH 6.7 and decreased by three- to fourfold at both acid and alkaline pH. TMO modification removed the pH dependence of channel kinetics, and average open times were short at all pHs between 5.5 and 8.1. We suggest that TMO modifies normally titratable groups on the external surface of ACh channels that help to determine both the gating and permeability properties of ACh channels.