Leishmania amazonensis infection may affect the ability of the host macrophage to be activated by altering their outward potassium currents

Understanding the impact of intracellular pathogens on the behaviour of their host cells is key to designing new interventions. We are interested in how Leishmania alters the electrical functioning of the plasma membrane of the macrophage it infects. The specific question addressed here is whether Leishmania amazonensis infection alters the macrophage’s outward currents and what the consequences of such changes might be. Using the whole cell configuration of the patch clamp technique, we show that outward peak current density remains constant over the period studied but that time to peak and sensitivity to inhibitors vary during infection. Infected cells take 40% longer to activate and are more sensitive to the potassium channel inhibitor tetraethyl ammonium, compared to control cells, indicating increased potassium outward current activity. Activation of macrophages is associated with increases of nitric oxide production and membrane area, depolarization of the macrophage membrane, down regulation of inward potassium and up regulation of outward currents. After Leishmania infection, macrophage activation is characterised by a reduction of nitric oxide production and of outward current density. We therefore suggest that this reflects a weaker activation.     

The relation of elevation of cytosolic free calcium to activation of membrane conductance in rat parotid acinar cells

The relation between elevation of cytosolic free calcium and activation of membrane conductance has been studied in single acinar cells of the rat parotid. Outward and inward currents are activated by calcium elevation and oscillate in phase with oscillations of cytosolic calcium. The outward current results from activation of a large unit-conductance Ca2+ and voltage-dependent K+ channel, whereas the inward current is most likely carried predominantly by Cl-. Both these conductances have been previously described in exocrine cells. Buffering calcium at resting levels eliminated current responses to muscarinic agonists, suggesting that calcium is the only significant second messenger involved in the short-term control of this conductance by acetylcholine.     

Membrane properties of isolated mudpuppy taste cells

The voltage-dependent currents of isolated Necturus lingual cells were studied using the whole-cell configuration of the patch-clamp technique. Nongustatory surface epithelial cells had only passive membrane properties. Small, spherical cells resembling basal cells responded to depolarizing voltage steps with predominantly outward K+ currents. Taste receptor cells generated both outward and inward currents in response to depolarizing voltage steps. Outward K+ currents activated at approximately 0 mV and increased almost linearly with increasing depolarization. The K+ current did not inactivate and was partially Ca++ dependent. One inward current activated at -40 mV, reached a peak at -20 mV, and rapidly inactivated. This transient inward current was blocked by tetrodotoxin (TTX), which indicates that it is an Na+ current. The other inward current activated at 0 mV, peaked at 30 mV, and slowly inactivated. This more sustained inward current had the kinetic and pharmacological properties of a slow Ca++ current. In addition, most taste cells had inwardly rectifying K+ currents. Sour taste stimuli (weak acids) decreased outward K+ currents and slightly reduced inward currents; bitter taste stimuli (quinine) reduced inward currents to a greater extent than outward currents. It is concluded that sour and bitter taste stimuli produce depolarizing receptor potentials, at least in part, by reducing the voltage-dependent K+ conductance.     

Changes in macrophage membrane properties during early Leishmania amazonensis infection differ from those observed during established infection and are partially explained by phagocytosis

Understanding the impact of intracellular pathogens on the behavior of their host cells is key to designing new interventions. We are interested in how Leishmania alters the electrical function of the plasma membrane of the macrophage it infects. The specific question addressed here is the impact of Leishmania infection on macrophage membrane properties during the first 12 h post-infection. A decrease of 29% in macrophage membrane capacitance at 3 h post-infection indicates that the phagolysosome membrane is donated on entry by the macrophage plasma membrane. Macrophage membrane potential depolarized during the first 12 h post-infection, which associated with a decreased inward potassium current density, changed in inward rectifier conductance and increased outward potassium current density. Decreased membrane capacitance and membrane potential, with no changes in ion current density, were found in macrophages after phagocytosis of latex beads. Therefore we suggest that the macrophage membrane changes observed during early Leishmania infection appear to be associated with the phagocytic and activation processes.     

Modulation of membrane potential and ionic currents by the AT1 and AT2 receptors of angiotensin II

Angiotensin II, the principal effector of the renin-angiotensin system, modulates various ionic currents. Its effects on potassium currents, including outward transient potassium current, the inward or outward rectifiers, as well as Ca²⁺-activated potassium currents, is well described. Other ionic currents, such as voltage-dependent calcium currents, cationic or chloride currents, are also altered by the hormone. All these effects provoke changes in membrane potential, such as modulation of action potential firing or resting membrane potential and control intracellular calcium concentration. Summarized here are the results obtained on these membrane electrical properties using electrophysiological recordings.     

Elevation of a potassium current in differentiating human leukemic (HL-60) cells

Human promyelocytic leukemia (HL-60) cells display a novel voltage-dependent outward current under voltage clamp. This current is present at low levels in the proliferative state and in granulocytes derived from HL-60 cells which were induced to differentiate with retinoic acid. It is elevated in macrophages derived from HL-60 cells after exposure to phorbol-12-myristate-13-acetate (PMA). The current is carried primarily by K+, is blocked by Cs+ and by increased intracellular concentrations of Cl-. From a holding potential of -80 mV, significant activation required depolarization to +20 mV membrane potential. Activation was not influenced by intracellular Ca2+ (1-2 X 10(-6) M). These properties appear to differ significantly from the Ca2+-activated K+ channel and the delayed rectifier. The increase of this voltage-activated current in differentiation toward the macrophage, but not the granulocyte, suggests that this current is correlated specifically with macrophage differentiation.     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

Voltage-dependent conductances in Limulus ventral photoreceptors

The voltage-dependent conductances of Limulus ventral photoreceptors have been investigated using a voltage-clamp technique. Depolarization in the dark induces inward and outward currents. The inward current is reduced by removing Na+ or Ca2+ and is abolished by removing both ions. These results suggest that both Na+ and Ca2+ carry voltage-dependent inward current. Inward current is insensitive to tetrodotoxin but is blocked by external Ni2+. The outward current has a large transient component that is followed by a smaller maintained component. Intracellular tetraethylammonium preferentially reduces the maintained component, and extracellular 4-amino pyridine preferentially reduces the transient component. Neither component is strongly affected by removal of extracellular Ca2+ or by intracellular injection of EGTA. It is concluded that the photoreceptors contain at least three separate voltage-dependent conductances: 1) a conductance giving rise to inward currents; 2) a delayed rectifier giving rise to maintained outward K+ current; and 3) a rapidly inactivating K+ conductance similar to the A current of molluscan neurons.     

Appearance and maturation of voltage-dependent conductances in solitary spiking cells during retinal regeneration in the adult newt

Summary Electrical membrane properties of solitary spiking cells during newt (Cynops pyrrhogaster) retinal regeneration were studied with whole-cell patch-clamp methods in comparison with those in the normal retina.The membrane currents of normal spiking cells consisted of 5 components: inward Na⁺ and Ca⁺⁺ currents and 3 outward K⁺ currents of tetraethylammonium (TEA)-sensitive, 4-aminopyridine (4-AP)-sensitive, and Ca⁺⁺-activated varieties. The resting potential was about -40mV. The activation voltage for Na⁺ and Ca⁺⁺ currents was about -30 and -17 mV, respectively. The maximum Na⁺ and Ca⁺⁺ currents were about 1057 and 179 pA, respectively.In regenerating retinae after 19–20 days of surgery, solitary cells with depigmented cytoplasm showed slowrising action potentials of long duration. The ionic dependence of this activity displayed two voltage-dependent components: slow inward Na⁺ and TEA-sensitive outward K⁺ currents. The maximum inward current (about 156 pA) was much smaller than that of the control. There was no indication of an inward Ca⁺⁺ current.During subsequent regeneration, the inward Ca⁺⁺ current appeared in most spiking cells, and the magnitude of the inward Na⁺, Ca⁺⁺, and outward K⁺ currents all increased. By 30 days of regeneration, the electrical activities of spiking cells became identical to those in the normal retina. No significant difference in the resting potential and the activation voltage for Na⁺ and Ca⁺⁺ currents was found during the regenerating period examined.     

A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.     

Whole cell K and Cl currents in dissociated eccrine secretory coil cells during stimulation

Using the whole-cell voltage clamp (to determine the membrane current) and current clamp (to determine membrane potential) methods in conjunction with the nystatin-perforation technique, we studied the effect of methacholine (MCh) and other secretagogues on whole cell K and Cl currents in dissociated rhesus palm eccrine sweat clear cells. Application of MCh by local superfusion induced a net outward current (at a holding potential of ?60 mV and a clamp voltage of 0 mV), and a transient hyperpolarization by 5.6 mV, suggesting the stimulation of K currents. The net outward current gradually changed to the inward (presumably Cl) currents over the next 1 to 2 min of continuous MCh stimulation. During this time the membrane potential also changed from hyperpolarization to depolarization. The inward currents were increasingly more activated than outward (presumably K) currents during repeated MCh stimulations so that a net inward current (at ?60 mV) was observed after the fourth or fifth MCh stimulation. Ionomycin (10 μm) also activated both inward and outward current. The observed effect of MCh was abolished by reducing extracellular [Ca] to below 1 nm (Ca-free + 1 mm EGTA in the bath). MCh-activated outward currents were inhibited by 5 mm Ba and by 0.1 mm quinidine, although these agents also suppressed the inward currents. Bi-ionic potential measurements indicated that the contribution of Na to the membrane potential was negligible both before and after MCh or ISO (isoproterenol) stimulations and that the observed membrane current was carried mainly by K and Cl. MCh increased the bi-ionic potential by step changes in external K and Cl concentrations, further supporting that MCh-induced outward and inward currents represent K and Cl currents, respectively. Stimulation with ISO or FK (forskolin) resulted in a depolarization by about 55 mV and a net inward (most likely Cl) current independent of external Ca. CT-cAMP mimicked the effects of FK and ISO. The bi-ionic potential, produced by step changes in the external Cl concentration, increased during ISO stimulation, whereas that of K decreased. This indicates that the ISO-induced inward current is due to Cl current and that K currents were unchanged or slightly decreased during stimulation with ISO or 10 μm FK. Both myoepithelial and dark cells responded only to MCh (but not to FK) with a marked depolarization of the membrane potential due to activation of Cl, but not K, currents. We conclude that MCh stimulates Ca-dependent K and Cl currents, whereas ISO stimulates cAMP-dependent Cl currents in eccrine clear cells.     

Tetrodotoxin-sensitive, voltage-dependent sodium currents in hair cells from the alligator cochlea.

We have used whole-cell patch clamp techniques to record from tall hair cells isolated from the apical half of the alligator cochlea. Some of these cells gave action potentials in response to depolarizing current injections. When the same cells were voltage clamped, large transient inward currents followed by smaller outward currents were seen in response to depolarizing steps. We studied the transient inward current after the outward current had been blocked by external tetraethylammonium (20 mM) or by replacing internal potassium with cesium. It was found to be a sodium current because it was abolished by either replacing external sodium with choline or by external application of tetrodotoxin (100 nM). The sodium current showed voltage-dependent activation and inactivation. Most of the spiking hair cells came from the apex of the cochlea, where they would be subject to low-frequency mechanical stimulation in vivo.     

Voltage-dependent calcium and calcium-activated potassium currents of a molluscan photoreceptor

Two-microelectrode voltage clamp studies were performed on the somata of Hermissenda Type B photoreceptors that had been isolated by axotomy from all synaptic interaction as well as any impulse-generating (i.e., active) membrane. In the presence of 2-10 mM 4-aminopyridine (4-AP) and 100 mM tetraethylammonium ion (TEA), which eliminated two previously described voltage-dependent potassium currents (IA and the delayed rectifier), a voltage-dependent outward current was apparent in the steady state responses to command voltage steps more positive than -40 mV (absolute). This current increased with increasing external Ca++. The magnitude of the outward current decreased and an inward current became apparent following EGTA injection. Substitution of external Ba++ for Ca++ also made the inward current more apparent. This inward current, which was almost eliminated after being exposed for approximately 5 min to a solution in which external Ca++ was replaced with Cd++, was maximally activated at approximately 0 mV. Elevation of external potassium allowed the calcium (ICa++) and calcium-dependent K+ (IC) currents to be substantially separated. Command pulses to 0 mV elicited maximal ICa++ but no IC because no K+ currents flowed at their new reversal potential (0 mV) in 300 mM K+. At a holding potential of -60 mV, which was now more negative than the potassium equilibrium potential, EK+, in 300 mM K+, IC appeared as an inward tail current after positive command steps. The voltage dependence of ICa++ was demonstrated with positive steps in 100 mM Ba++, 4-AP, and TEA. Other data indicated that in 10 mM Ca++, IC underwent pronounced and prolonged inactivation whereas ICa++ did not. When the photoreceptor was stimulated with a light step (with the membrane potential held at -60 mV), there was also a prolonged inactivation of IC. In elevated external Ca++, ICa++ also showed similar inactivation. These data suggest that IC may undergo prolonged inactivation due to a direct effect of elevated intracellular Ca++, as was previously shown for a voltage-dependent potassium current, IA. These results are discussed in relation to the production of training-induced changes of membrane currents on retention days of associative learning.     

Whole-Cell K+ Currents across the Plasma Membrane of Tobacco Protoplasts from Cell-Suspension Cultures

The whole-cell configuration of the patch clamp technique was used to study both outward and inward ion currents across the plasma membrane of tobacco (Nicotiana tabacum) protoplasts from cell-suspension cultures. The ion currents across the plasma membrane were analyzed by the application of stepwise potential changes from a holding potential or voltage ramps. In all protoplasts, a voltage- and time-dependent outward rectifying current was present. The conductance increased upon depolarization of the membrane potential (to >0 mV) with a sigmoidal time course. The reversal potential of the outward current shifted in the direction of the K+ equilibrium potential upon changing the external K+ concentration. The outward current did not show inactivation. In addition to the outward rectifying current, in about 30% of the protoplasts, a time- and voltage-dependent inward rectifying current was present as well. The inward rectifying current activated upon hyperpolarization of the membrane potential (<-100 mV) with an exponential time course. The reversal potential of the inward conductance under different ionic conditions was close to the K+ equilibrium potential.     

Electrical properties and transmembrane ion currents of single smooth-muscle cells

Current and voltage clamp investigations of freshly isolated smooth muscle cells from guinea-pig ileum and taenia coli were performed using single suction micropipette technique. Specific membrane capacity of smooth muscle cells was calculated and accounted for 1.6 microF/cm2, with specific resistance varying from 50 to 150 k omega X cm2. Transmembrane currents consisted of two inward components, inactivating and noninactivating ones, carried by Ca2+ ions, overlapping with early activated potassium outward current. Time constant of inward current activation was not only voltage-sensitive but also ion-dependent. When Ca2+ ions in Krebs solution were replaced by Ba2+, both the rate of activation and inactivation of inward current were significantly reduced. Estimation of intracellular Ca2+ concentration increase has indicated that inward calcium current transports enough Ca2+ for direct contraction activation.     

Investigation of the TEA-resistant outward current in the somatic membrane of perfused nerve cells

Outward currents remaining after addition of 20–50 mM of tetraethylammonium (TEA) ions to the extracellular or intracellular solution, were investigated in perfused isolatedHelix neurons. After this addition, the inactivated inward current carried by potassium ions, the potential-dependent and kinetic characteristics of which differ from those of potassium outward currents suppressed by TEA, is preserved in the membrane. A component dependent on the inward calcium current was found in this TEA-resistant outward current; it was abolished by replacement of the extra-cellular calcium ions by magnesium ions, by blocking of the calcium channels by extracellular cadmium ions, and by their destruction by intracellular fluoride ions. Increasing the intracellular concentration of free calcium ions by perfusing the cell with solutions containing calcium-EGTA buffer potentiated the TEA-resistant component of the outward current, whereas removal of these ions with EGTA weakened it. It is concluded that a system of outward current channels whose activation depends on the presence of calcium ions near the inner surface of the membrane is present in the somatic membrane. It is suggested that to keep these channels capable of being activated, calcium ions must bind with the structures forming their internal opening.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 460–468, September–October, 1979.     

Bradykinin inhibits potassium (M) currents in N1E-115 neuroblastoma cells. Responses resemble those in NG108-15 neuroblastoma x glioma hybrid cells

Application of bradykinin to voltage-clamped N1E-115 mouse neuroblastoma cells evoked sequential outward and inward membrane currents, accompanied by an increase and decrease of membrane conductance, respectively. Methacholine produced an inward current with a decreased conductance. The outward current response to bradykinin was imitated by intracellular inositol 1,4,5-trisphosphate (IP3). Bath application of phorbol dibutyrate induced an inward current and potentiated the response to IP3. We conclude that the response of these cells to bradykinin is identical to that of NG108-15 hybrid cells, and therefore may be attributed to the dual effects of inositol trisphosphate and diacylglycerol formed by hydrolysis of phosphatidylinositide.     

睫状体色素上皮细胞容积激活性氯电流

为研究睫状体色素上皮 (pigmentedciliaryepithelial,PCE)细胞容积激活性Cl-电流的特性 ,用膜片箝全细胞记录技术记录了猪的低渗液诱发的容积激活性Cl-电流。此电流外向占优势 ,几乎没有时间依赖性失活 ,电流 电压曲线显示此电流反转电位 (- 6 3± 0 5mV)很接近氯离子平衡电位的计算值 (ECl=0mV)。电流的激活依赖于细胞内ATP ,细胞外ATP抑制外向电流和内向电流 ,但外向电流抑制率大于内向电流抑制率 (92 %比 74% ,P <0 0 1)。氯离子通道阻断剂tamoxifen抑制外向电流和内向电流 ,两个抑制率几乎相等 (85 %比 87% ,P >0 0 5 )。此电流特性与其他类型细胞的P糖蛋白相关电流很相似。结果提示PCE细胞容积激活性Cl-电流的形成可能与P糖蛋白有关     

Differential voltage-dependent K+ channel responses during proliferation and activation in macrophages

Voltage-dependent K+ channels (VDPC) are expressed in most mammalian cells and involved in the proliferation and activation of lymphocytes. However, the role of VDPC in macrophage responses is not well established. This study was undertaken to characterize VDPC in macrophages and determine their physiological role during proliferation and activation. Macrophages proliferate until an endotoxic shock halts cell growth and they become activated. By inducing a schedule that is similar to the physiological pattern, we have identified the VDPC in non-transformed bone marrow-derived macrophages and studied their regulation. Patch clamp studies demonstrated that cells expressed outward delayed and inwardly rectifying K+ currents. Pharmacological data, mRNA, and protein analysis suggest that these currents were mainly mediated by Kv1.3 and Kir2.1 channels. Macrophage colony-stimulating factor-dependent proliferation induced both channels. Lipopolysaccharide (LPS)-induced activation differentially regulated VDPC expression. While Kv1.3 was further induced, Kir2.1 was down-regulated. TNF-alpha mimicked LPS effects, and studies with TNF-alpha receptor I/II double knockout mice demonstrated that LPS regulation mediates such expression by TNF-alpha-dependent and -independent mechanisms. This modulation was dependent on mRNA and protein synthesis. In addition, bone marrow-derived macrophages expressed Kv1.5 mRNA with no apparent regulation. VDPC activities seem to play a critical role during proliferation and activation because not only cell growth, but also inducible nitric-oxide synthase expression were inhibited by blocking their activities. Taken together, our results demonstrate that the differential regulation of VDPC is crucial in intracellular signals determining the specific macrophage response.