Control of membrane potential by external H concentration in Bacillus subtilis as determined by an ion-selective electrode

The membrane potential of intact bacteria was monitored by measuring the tetraphenylphosphonium ion distribution across the membrane using poly-(vinyl chloride) matrix-type electrode selective to tetraphenylphosphonium ion. It was found that the tetraphenylphosphonium ion was not countertransported against H⁺ movement. The membrane potential of Bacillus subtilis was estimated to be 80–120 mV inside-negative at external pH 7. The effect of the external pH on the membrane potential was studied. It varied from 30 to 40 mV/decade change in the external [H⁺] in the pH region of greater than 6.5, increasing pH making it more inside-negative. The addition of carbonyl cyanide m-chlorophenylhydrazone depolarized the membrane, and the membrane potential approached the H⁺ equilibrium potential. The addition of N,N′-dicyclohexylcarbodiimide did not abolish the pH dependence of the membrane potential. Increasing the external [K⁺] did not affect the pH dependence. CN⁻ partially depolarized the membrane. A parallel conductance model for membrane potential could explain the results qualitatively.     

Interactive signal transfer between host and pathogen during successful infection of barley leaves by Blumeria graminis and Bipolaris sorokiniana

Using ion-selective microprobes, interactive signalling between barley and Blumeria graminis or Bipolaris sorokiniana has been investigated. The question was raised whether a biotrophically growing fungus manipulates the electrical driving forces (membrane potential, transmembrane pH), required for H⁺ cotransport of energy-rich compounds. Electrodes were positioned in the substomatal cavity of open stomata or on the leaf surface, and pH was measured continuously up to several days during fungal development. We demonstrate that surface and apoplastic fluids are electrically coupled and respond in a similar manner to stimuli. Apoplastic pH, monitored from the moment of inoculation with conidia, reveals several phases: 2–4 h after inoculation of the barley leaf with either fungus, the host displays rapid transient responses after its first contact with the fungal cell wall; apoplastic pH and pCa increases, cytoplasmic pH and pCa decreases. About 1 day after inoculation, the apoplastic pH increases by up to 2 pH units, which is thought to reflect a resistance response against the intruder. Whereas barley leaf cells possess a membrane potential of −152±5 mV, hyphae of B. graminis yield −251±8 mV, indicative of a substantial driving force advantage for the fungus. Although the resting membrane potential of barley remains constant during the first days after inoculation, leaves infected with B. sorokiniana get confronted with an energy problem, indicated by a retarded repolarization following a “light-off” stimulus. Five days after inoculation, apoplastic pH has increased to 5.97±0.47 (n=11) and does no longer respond to “light-off” when measured within lesions. In contrast, it stays at near normal values outside the lesions and responds to “light-off”.It is concluded that biotrophically growing fungi do not manipulate the cotransport driving forces since (i) any change in apoplastic pH would be experienced by both partners; (ii) the resting membrane potential is not changed. It is suggested that measured pH changes reflect defence responses of the host against the fungus rather than fungal action to increase compatibility.     

"Action potentials" in Neurospora crassa, a mycelial fungus.

Occasional spontaneous "action potentials" are found in mature hyphae of the fungus Neurospora crassa. They can arise either from low-level sinusoidal oscillations of the membrane potential or from a linear slow depolarization which accelerates into a rapid upstroke at a voltage 5-20 mV depolarized from the normal resting potential (near-180 mV). The "action potentials" are long-lasting, 1-2 min and at the peak reach a membrane potential near-40 mV. A 2-to 8-fold increase of membrane conductance accompanies the main depolarization, but a slight decrease of membrane conductance occurs during the slow depolarization. Two plausible mechanisms for the phenomenon are (a) periodic increases of membrane permeability to inorganic ions, particularly H+ or Cl- and (b) periodic decreases in activity of the major electrogenic pump (H+) or the Neurospora membrane, coupled with a nonlinear (inverse signoid) current-boltage relationship. Identification of action potential-like disturbances in fungi means that such behavior has now been found in all major biologic taxa which have been probed with suitable electrodes. As yet there is no obvious function for the events in fungi.     

Acid dissociation constants and pH values for standard “bes” and “tricine” buffer solutions in 30, 40, and 50 mass% dimethyl sulfoxide/water between 25 and −25 °C

Information is given concerning two standard buffer solutions suitable as pH references in 30, 40, and 50 mass% dimethyl sulfoxide (DMSO)/H₂O mixed solvents at subzero temperatures from −20 to 0 °C, with the intention of establishing a pH (designated pH^*) scale. The two buffers selected were the ampholytes N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid (“bes”) and N-tris(hydroxymethyl)methylglycine (“tricine”), and the reference standard consisted of equal molal quantities of the buffer and its respective sodium salt. The assignment of pH^* values was based on measurements of the emf of cells without liquid junction of the type: Pt;H₂(g,1 atm) ¦Bes, Na Besate, NaCl ¦ AgCl;Ag and Pt;H₂(g,1 atm) ¦Tricine, Na Tricinate, NaCl ¦AgCl;Ag and the pH^* was derived from a determination of K₂, the equilibrium constant for the dissociation process (Buffer)^±/ai (Buffer)⁻ + H⁺.     

Volume-sensitive Cl<Superscript>−</Superscript> Current in Bovine Adrenocortical Cells: Comparison with the ACTH-induced Cl<Superscript>−</Superscript> Current

In a previous study performed on zona fasciculata (ZF) cells isolated from calf adrenal glands, we identified an ACTH-induced Cl⁻ current involved in cell membrane depolarization. In the present work, we describe a volume-sensitive Cl⁻ current and compare it with the ACTH-activated Cl⁻ current. Experiments were performed using the whole-cell patch-clamp recording method, video microscopy and cortisol-secretion measurements. In current-clamp experiments, hypotonic solutions induced a membrane depolarization to −22 mV. This depolarization, correlated with an increase in the membrane conductance, was sensitive to different Cl⁻ channel inhibitors. In voltage-clamp experiments, hypotonic solution induced a membrane current that slowly decayed and reversed at −21 mV. This ionic current displayed no time dependence and showed a slight outward rectification. It was blocked to variable extent by different conventional Cl⁻-channel inhibitors. Under hypotonic conditions, membrane depolarizations were preceded by an increase in cell volume that was not detected under ACTH stimulation. It was concluded that hypotonic solution induced cell swelling, which activated a Cl⁻ current involved in membrane depolarization. Although cell volume change was not observed in the presence of ACTH, biophysical properties and pharmacological profile of the volume-sensitive Cl⁻ current present obvious similarities with the ACTH-activated Cl⁻ current. As compared to ACTH, hypotonic solutions failed to trigger cortisol production that was weakly stimulated in the presence of high-K⁺ solution. This shows that in ZF cells, membrane depolarization is not a sufficient condition to fully activate secretory activities.This revised version was published online in August 2005 with a corrected cover date.     

TheChara plasmalemma at high pH. Electrical measurements show rapid specific passive uniport of H+ or OH−

Summary Above a critical external pH (about 10.5), theChara membrane acquires new propertes. In this state the membrane potential is close to the equilibrium potentials for H⁺ and OH^–, hyperpolarizing as external pH increases with a slope of –59 mV/pH unit. The membrane conductance increases by an average factor of 2.4 above the critical pH. These changes are explained by an increase in permeability to OH^– (or H⁺). The establishment of a OH^– (or H⁺ permeable membrane at high pH suggests that the large fluxes of OH^– (or H⁺ which occur in the alkaline band in photosynthesizing cells are passive.     

Growth and functional responses of different cultivars of Lotus corniculatus to aluminum and low pH stress

Aluminum toxicity is an important stress factor in acid soils. Growth, respiration and permeability properties of root cells were studied in five cultivars of Lotus corniculatus subjected to aluminum (Al) or low pH stress. The cultivars showed significant differences in root elongation under stress conditions, which correlated with changes in membrane potential (E_M) of root cortical cells. A pH drop from 5.5 to 4.0 resulted in significant membrane depolarization and root growth inhibition. The strongest inhibition was observed in cv. São Gabriel (33.6%) and least in cv. UFRGS (25.8%). Application of an extremely high Al concentration (2 mM) stopped the root growth in cv. INIA Draco, while inhibition in cv. UFRGS reached only 75%.The E_M values of cortical cells of Lotus roots varied between −115 and −144 mV. Treatment with 250 μM of AlCl₃ (pH 4) resulted in rapid membrane depolarization. The extent of the membrane depolarization ranged between 51 mV (cv. UFGRS) and 16 mV (cv. INIA Draco). The membrane depolarization was followed by a loss of K⁺ from Al-treated roots (2 mM Al) and resulted in a decrease of the diffusion potential (E_D). The total amount of K⁺ in Al-treated roots dropped from 31.4 to 16.8 μmol g⁻¹ FW in sensitive cv. INIA Draco, or from 26.1 to 22.7 μmol g⁻¹ FW in tolerant cv. UFGRS. The rate of root respiration under control conditions as well as under Al treatment was higher in cv. INIA Draco than in cv. UFRGS. Al-induced inhibition of root respiration was 21–34% of the control.     

Cyanine dye fluorescence used to measure membrane potential changes due to the assembly of complement proteins C5b-9

Summary The fluorescent potentiometric indicator diS–C₃-(5) has been used to investigate changes in membrane potential due to assembly of the C5b-9 membrane attack complex of the complement system. EAC1-7 human red blood cells and resealed erythrocyte ghosts—bearing membrane-assembled C5b67 complexes—were generated by immune activation in C8-deficient human serum. Studies performed with these cellular intermediates revealed that the membrane potential of EAC1-7 red cells and ghosts is unchanged from control red cells (–7 mV) and ghosts (0 mV), respectively. Addition of complement proteins C8 and C9 to EAC1-7 red cells results in a dose-dependent depolarization of membrane potential which precedes hemolysis. This prelytic depolarization of membrane potential—and the consequent onset of hemolysis—is accelerated by raising external [K⁺], suggesting that the diffusional equilibration of transmembrane cation gradients is rate limiting to the cytolytic event. In the case of EAC1-7 resealed ghosts suspended at either high external [K⁺] or [Na⁺], no change in membrane potential (from 0 mV) could be detected after C8/C9 additions. When the membrane potential of the EAC1-7 ghost was displaced from 0 mV by selectively increasing the K⁺ conductance with valinomycin, a dose-dependent depolarization of the membrane was observed upon addition of C8 and C9. In these experiments, lytic breakdown of the ghost membranes was <5%. Conclusions derived from this study include: (i) measured prelytic depolarization of the red cell Donnan potential directly confirms the colloid-osmotic theory of immune cytolysis. (ii) The diffusional transmenbrane equilibration of Na⁺ and K⁺ through the C5b-9 pore results in a dose-dependent depolarization of the membrane potential (E _m ) which appears to be rate-limiting to cytolytic rupture of the target erythrocyte. (iii) Enhanced immune hemolysis observed in high K⁺ media cannot be attributed to cation-selective conductance across the C5b-9 pore, and is probably related to the nearequilibrium condition of potassium-containing red cells when suspended at high external K⁺. These experiments demonstrate that carbocyanine dye fluorescent indicators can be used to monitor electrochemical changes arising from immune damage to the plasma membrane under both cytolytic and noncytolytic conditions. Potential application of this method to the detection of sublytic pathophysiological changes in the plasma membrane of complement-damaged cells are discussed.     

Evidence for a simple Cl conductance pathway in nutrient membrane of frog stomach

A decrease in nutrient Cl⁻ results in an increased negativity of the nutrient relative to the secretory side. The possibility emerged that Cl⁻ transport could be attributed to a neutral mechanism involving Cl⁻ in the nutrient membrane coupled to a simple Cl⁻ conductance pathway in the secretory membrane. The decrease in PD (potential difference) with a decrease in nutrient Cl⁻ could arise from a decrease in cellular Cl⁻ so that the ratio of Cl⁻ in cell to Cl⁻ in secretory solution was decreased. Experiments were designed to determine whether there was a need to assume a simple Cl⁻ conductance pathway. A 10-fold decrease in Cl⁻ gave in HCO₃⁻-containing nutrient solutions a PD decrease of 20 mV, in HCO₃⁻-free nutrient solutions, a PD decrease of 13.5 mV, and in HCO₃⁻-free and Na⁺-free solutions, a PD decrease of 6.7 mV. The decrease of 6.7 mV could not be attributed to a neutral Cl⁻HCO₃⁻ exchanger or a NaCl symport. Also there was no evidence for a KCl symport from changes in Cl⁻ in presence and absence of K⁺. It followed that the decrease of 6.7 mV provided evidence for a simple Cl⁻ conductance pathway in the nutrient membrane.     

Studies on the perfused plasmalemma ofChara corallina: I. Current-voltage curves: ATP and potassium dependence

Summary The electrical properties of theChara cell membrane have been studied using a perfusion method based on that of Williamson, R.E. 1975.J. Cell Sci. 17655. The vacuole, tonoplast, and inner cytoplasm are removed by a brief rapid perfusion. Electrical properties of the plasmalemma indicate that it remains intact after this perfusion.The membrane potential difference after perfusion and with no ATP was close to the potassium equilibrium potential; the current-voltage characteristic had a slope that was time- and voltage-dependent, indicating that the steady-state potassium conductance increased with depolarization. At –125 mV the membrane conductance of the plasmalemma depended on [K⁺]₀. This dependence was inhibited by perfusing with 2.0mm ATP or by clamping at a more negative membrane potential. The addition of ATP to the perfusion medium of unclamped cells caused a hyperpolarization ofca. 50 mV, presumably by activating the proton pump. In clamped cells, perfusion with ATP caused currents ofca. 20 mA m^–2, whose magnitude depended on pH₀. ATP induced membrane conductance changes which were variable. 2.0mm ADP inhibited the proton pump. The intersection points of current-voltage characteristics can set limits on the stalling potential; the resulting stoichiometry of the proton pump appears to be 1.5–2.0 H⁺ per ATP.     

Electrophysiological studies of forskolin-induced changes in ion transport in the human colon carcinoma cell line HT-29 cl.19A: Lack of evidence for a cAMP-activated basolateral K+ conductance

Summary Forskolin (i.e, cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl^–-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments.Changes in electrophysiological parameters provoked by serosal addition of 10^–5 m forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9±0.4 mV). (ii) a transient decrease in transepithelial resistance with 26±3 · cm² from a mean value of 138±13 · cm² before forskolin addition, (iii) a depolarization of the cell membrane potential by 24±1 mV from a resting value of –50±1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80±0.02 to 0.22±0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl^– in the medium. Subsequent addition of bumetanide (10^–4 m), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba²⁺ (1mm), a known inhibitor of basolateral K⁺ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K⁺ conductance.The results suggest that forskolin-induced Cl^– secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl^– conductance of the apical membrane but does not affect K⁺ or Cl^– conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl^– and the basal Ba²⁺-sensitive K⁺ conductance are sufficiently large to maintain the Cl^– efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl^– channel blockers NPPB and DPC on basolateral conductance rather than apical Cl^– conductance.     

Potassium-proton symport inNeurospora: kinetic control by pH and membrane potential

Summary Active transport of potassium in K⁺-starvedNeurospora was previously shown to resemble closely potassium uptake in yeast,Chlorella, and higher plants, for which K⁺ pumps or K⁺/H⁺-ATPases had been proposed. ForNeurospora, however, potassium-proton cotransport was demonstrated to operate, with a coupling ratio of 1 H⁺ to 1 K⁺ taken inward so that K⁺, but not H⁺, moves against its electrochemical gradient (Rodriguez-Navarro et al.,J. Gen. Physiol. 87:649–674).In the present experiments, the current-voltage (I–V) characteristic of K⁺–H⁺ cotransport in spherical cells ofNeurospora has been studied with a voltage-clamp technique, using difference-current methods to dissect it from other ion-transport processes in theNeurospora plasma membrane. Addition of 5-200 M K⁺ to the bathing medium causes 10–150 mV depolarization of the unclamped membrane, and yields a sigmoidI–V curve with a steep slope (maximal conductance of 10–30 S/cm²) for voltages of –300 to –100 mV, i.e., in the normal physiologic range. Outside that range the apparentI–V curve of the K⁺-H⁺ symport saturates for both hyperpolarization and depolarization. It fails to cross the voltage axis at its predicted reversal potential, however, an effect which can be attributed to failure of theI–V difference method under reversing conditions.In the absence of voltage clamping, inhibitors—such as cyanide or vanadate—which block the primary proton pump inNeurospora also promptly inhibit K⁺ transport and K⁺-H⁺ currents. But when voltage clamping is used to offset the depolarizing effects of pump blockade, the inhibitors have no immediate effect on K⁺-H⁺ currents. Thus, the inhibition of K⁺ transport usually observed with these agents reflects the kinetic effect of membrane depolarization rather than any direct chemical action on the cotransport system itself.Detailed study of the effects of [K⁺]_o and pH_o on theI–V curve for K⁺-H⁺ symport has revealed that increasing membrane potential systematicallydecreases the apparent affinity of the transporter for K⁺, butincreases affinity for protons (K _m range: for [K⁺]_o, 15–45 M; for [H⁺]_o, 10–35 nM). This behavior is consistent with two distinct reaction-kinetic models, in which (i) a neutral carrier binds K⁺ first and H⁺ last in the forward direction of transport, or (ii) a negatively charged carrier (–2) binds H⁺ first and K⁺ last.     

The acetylcholine receptor: Isolation of a brain nicotinic receptor and its preliminary characterization in lipid bilayer membranes

The technique of affinity chromatography with the curarizing neurotoxins of Naja naja venom has been employed to extract nicotinic acetylcholine receptors from the brain tissues of mouse and hog. Both carbochol and hexamethonium were used as linear or step gradients to elute the receptor and its properties were investigated in lipid bilayer membranes. Of particular interest is the observation that discrete quanta of conductance could be observed across an NaCl gradient of 1.0:0.1 M. By switching the voltage-clamp across the bilayer between a positive and negative 80 mV, the separate Na⁺ and Cl⁻ conductances of these quanta could be estimated and the following conductances of the smallest discrete quanta were observed: 3.7 · 10⁻¹¹ Ω⁻¹ (Na⁺) and 5.9 · 10⁻¹¹ Ω⁻¹ (Cl⁻) for mouse brain receptors; 3.8 · 10⁻¹¹ Ω⁻¹ (Na⁺) and 4.7 · 10⁻¹¹ Ω⁻¹ (Cl⁻) for hog brain receptors. Large aggregates of receptors appeared to activate and deactivate as multiples of a basic conductance size, although there is evidence that they may not represent the actual gating of ion channels. A “background noise” that is not within the temporal capability of the recording system is also present at an intensity that seems to parallel the number of activated receptors, and in view of recent electrophysiological evidence that the relaxation lifetime of the open channel state is of a millisecond duration, it may be that this “noise” actually represent the channel gating.     

Electrophysiological and pharmacological properties of cultured rat thyroid cells

The electrophysiological properties of a hormone-dependent, differentiated thyroid epithelial cell strain were studied using intracellular microelectrodes. The average membrane potential of solitary, isolated cells was –78.4 ± 1.3 mV. The membrane potential depolarized 55 mV per tenfold increase in extracellular potassium concentation. Weak electrical coupling was recorded between contiguous cells. Like tyroid cells in vivo, these cells did not generate action potentials. In some cells a spontaneous, slow transition in the membrane potential from –80mV to –30 mV was accompanied by an increase in input resistance. Membrane potential transitions could be induced by perfusing cells with isotonic Hanks solutions saturated with CO₂ (pH = 5.5) or by perfusing cells with hypotonic Hanks solutions (190–290 mOsm/kg). Membrane potential transitions were due to a decreased potassium permeability. Noradrenaline elicted both a fast depolarization and a slow depolarization. The fast depolarization was due to an increase in conductance of Na⁺ channels and of Cl^– channels. Intracellular injection of Ca⁺⁺ elicited the fast depolarization. Intracellular injection of EGTA or cobalt abolished the fast depolarization. Replacemnt of extracellular Ca⁺⁺ by Mg⁺⁺ did not affect the fast depolarization. Thus, the fast depolarization was due to accumulation of intracellular Ca⁺⁺. The fast depolarization was abolished by the alpha adrenergic blocker phentolamine (10^–6 M), and was not abolished by the beta adrenergic blocker propranolol (10^–5 M).     

High-conductance K+ channel in pancreatic islet cells can be activated and inactivated by internal calcium

Summary The Ca²⁺-activated K⁺ channel in rat pancreatic islet cells has been studied using patch-clamp single-channel current recording in excised inside-out and outside-out membrane patches. In membrane patches exposed to quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) large outward current steps were observed when the membrane was depolarized. The single-channel current voltage (I/V) relationship showed outward rectification and the null potential was more negative than –40 mV. In symmetrical K⁺-rich solutions the single-channelI/V relationship was linear, the null potential was 0 mV and the singlechannel conductance was about 250 pS. Membrane depolarization evoked channel opening also when the inside of the membrane was exposed to a Ca²⁺-free solution containing 2mm EGTA, but large positive membrane potentials (70 to 80 mV) were required in order to obtain open-state probabilities (P) above 0.1. Raising the free Ca²⁺ concentration in contact with the membrane inside ([Ca²⁺]_i) to 1.5×10^–7 m had little effect on the relationship between membrane potential andP. When [Ca²⁺]_i was increased to 3×10^–7 m and 6×10^–7 m smaller potential changes were required to open the channels. Increasing [Ca²⁺]_i further to 8×10^–7 m again activated the channels, but the relationship between membrane potential andP was complex. Changing the membrane potential from –50 mV to +20 mV increasedP from near 0 to 0.6 but further polarization to +50 mV decreasedP to about 0.2. The pattern of voltage activation and inactivation was even more pronounced at [Ca²⁺]_i=1 and 2 m. In this situation a membrane potential change from –70 to +20 mV increasedP from near 0 to about 0.7 but further polarization to +80 mV reducedP to less than 0.1. The high-conductance K⁺ channel in rat pancreatic islet cells is remarkably sensitive to changes in [Ca²⁺]_i within the range 0.1 to 1 m which suggests a physiological role for this channel in regulating the membrane potential and Ca²⁺ influx through voltage-activated Ca²⁺ channels.     

Spontaneous inactivation of the Ca-sensitive K channels of human red cells at high intracellular Ca activity

Ionophore A23187-mediated Ca²⁺-induced oscillations in the conductance of the Ca²⁺-sensitive K⁺ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K⁺ activity were followed with a K⁺-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular ⁴⁵Ca content. An increased cellular ⁴⁵Ca content at the successive minima of the oscillations where the K⁺ channels are closed indicates that the activation of the channels might be a (dCa²⁺/dt)-sensitive process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K⁺ channels at intracellular ionized calcium levels of about 10 μM and a concurrent membrane potential of about −65 mV was observed. At a membrane potential of about −70 mV and an intracellular concentration of about 2·10⁻⁴M no inactivation of K⁺ channels took place. Inactivation of the K⁺ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular ⁴⁵Ca content support the notion that a necessary component of the oscillatory system is a Ca²⁺ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium.     

Measurement of membrane potential in polymorphonuclear leukocytes and its changes during surface stimulation

The membrane potential of guinea pig polymorphonuclear leukocytes has been assessed with two indirect probes, tetraphenylphosphonium (TPP⁺) and 3,3′-dipropylthiadicarbocyanine (diS-C₃-(5)). The change in TPP⁺ concentration in the medium was measured with a TPP⁺-selective electrode. By monitoring differences in accumulation of TPP⁺ in media containing low and high potassium concentrations, a resting potential of −58.3 mV was calculated. This potential is composed of a diffusion potential due to the gradient of potassium, established by the Na⁺, K⁺ pump, and an electrogenic potential. The chemotactic peptide fMet-Leu-Phe elicits a rapid efflux of accumulated TPP⁺ (indicative of depolarization) followed by its reaccumulation (indicative of repolarization). In contrast, stimulation with concanavalin A results in a rapid and sustained depolarization without a subsequent repolarization. The results obtained with TPP⁺ and diS-C₃-(5) were comparable. Such changes in membrane potential were observed in the absence of extracellular sodium, indicating that an inward movement of sodium is not responsible for the depolarization. Increasing potassium levels, which lead to membrane depolarization, had no effect on the oxidative metabolism in nonstimulated or in fMet-Leu-Phe-stimulated cells. Therefore, it seems unlikely that membrane depolarization per se is the immediate stimulus for the respiratory burst.     

Membrane potential, action potential and activation potential of eggs of the sea urchin, Lytechinus variegatus

Unfertilized Lytechinus variegatus eggs in sea water in their normal physiological state have membrane potentials that approximate ?70 to ?80 mV. This conclusion is based on microelectrode measurements and on computation from the Na⁺ and K⁺ fluxes. The ?8 to ?15 mV values for the membrane potential previously reported and which are generally measured are the consequence of depolarization by impalement. The activation potential in inseminated eggs with an initial membrane potential more negative than ?60 mV is a compound event involving sperm-induced as well as voltage dependent conductance changes. The sperm-induced mechanism is a two-phase conductance increase which involves both Na⁺ and Ca²⁺ during the first phase, and Na⁺ alone during the second phase. In addition, the sperm-induced depolarization at the beginning of the first phase activates a voltage dependent Ca²⁺-conductance mechanism resulting in generation of an action potential.     

Suppression of the plasma membrane H<Superscript>+</Superscript>-conductance on the background of high H<Superscript>+</Superscript>-pump activity in dithiothreitol-treated <Emphasis Type="Italic">Chara</Emphasis> cells

Photosynthesizing cells of characean algae exposed to light are able to produce pH bands corresponding to alternate areas with dominant H⁺-pump activity and high H⁺-conductance of the cell membrane. The action potential generation temporally arrests the counter-directed H⁺ fluxes, which gives rise to opposite pH shifts in different cell regions and represents a suitable indicator for activities of the plasma membrane H⁺-transporting systems. Measurements of pH near the cell surface by means of microelectrodes and microspectrophotometry in the presence of pH-indicating dye thymol blue have shown that the treatment of cells with dithiothreitol (SH-group reducing agent) suppresses pH changes induced by the action potential generation in the alkaline cell areas and considerably increases the concurrent pH changes in the acid regions. Measurements of plasma membrane resistance in the alkaline zones revealed that dithiothreitol inhibits the light-dependent conductance of the resting cell and diminishes the conductance inactivation caused by the action potential generation. The data suggest that the reduction of accessible disulfide bonds results in the decrease of H⁺-conductance, whereas the activity of plasma membrane H⁺-pump remains unimpaired or is even enhanced.     

Voltage-activated ionic channels and conductances in embryonic chick osteoblast cultures

Summary Patch-clamp measurements were made on osteoblast-like cells isolated from embryonic chick calvaria. Cell-attachedpatch measurements revealed two types of high conductance (100–250 pS) channels, which rapidly activated upon 50–100 mV depolarization. One type showed sustained and the other transient activation over a 10-sec period of depolarization. The single-channel conductances of these channel types were about 100 or 250 pS, depending on whether the pipettes were filled with a low K⁺ (3mm) or high K⁺ (143mm) saline, respectively. The different reversal potentials under these conditions were consistent with at least K⁺ conduction. Whole-cell measurements revealed the existence of two types of outward rectifying conductances. The first type conducts K⁺ ions and activates within 20–200 msec (depending on the stimulus) upon depolarizing voltage steps from <–60 mV to >–30 mV. It inactivates almost completely with a time constant of 2–3 sec. Recovery from inactivation is biphasic with an initial rapid phase (1–2 sec) followed by a slow phase (>20 sec). The second whole-cell conductance activates at positive membrane potentials of >+50 mV. It also rapidly turns on upon depolarizing voltage steps. Activation may partly disappear at the higher voltages. Its single channels of 140 pS conductance were identified in the whole cell and did conduct K⁺ ions but were not highly Cl^– or Na⁺ selective. The results show that osteoblasts may express various types of voltage controlled ionic channels. We predict a role for such channels in mineral metabolism of bone tissue and its control by osteoblasts.