Electroimmunology: the physiologic role of ion channels in the immune system (original) (raw)
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Biochemical and Biophysical Research Communications, 1989
Bretylium tosylate -a sodium channel opener -resulted in an increase of membrane potential of depolarized human, rat and mouse T and B lymphocytes. Flow cytometric membrane potential measurements with bis-oxonol revealed that the above hyperpolarizing effect was amiloride, ouabain, tetrodotoxin, azide and temperature sensitive. The effect showed an absolute dependen~ on the extracellular sodium but it was insensitive to the extracellular Ca ~level. The voltage gating of the effect can be eliminated by either an increase of the extracellular potassium concentration or low doses of veratrin. The existence of a voltage and ligand gated sodium channel is suggested in the plasma membrane of all kinds of lymphocytes. The hyperpolarization is explained by an increased activity of the electrogenic sodium-potassium ATP-ase. Induced opening of such sodium channels may regulate the electrogenic pump activity and indirectly cell activation.
Voltage-dependent ion channels in T-lymphocytes
Journal of Neuroimmunology, 1985
accounted for without calcium channels. In particular, we show that many of the effects of 'calcium channel antagonists' such as verapamil, nifedipine, diltiazem and some polyvalent cations, can be accounted for by their blocking of voltage-gated potassium channels.
High-throughput profiling of ion channel activity in primary human lymphocytes
2008
This document contains the following supporting information for the article: • Supporting Methods • Table S-1: Throughput of automated ion channel assay for lymphocyte subsets. • Figure S-1: Confirming full blockage of Kv1.3 ion channels • Figure S-2: Specificity of the high-throughput electrophysiology method for Kv1.3 ion channel activity • Figure S-3: Algorithms to quantify Kv1.3-specific currents • Figure S-4: Individual time-courses of functional Kv1.3 activity after mitogenic stimulation in three different subjects • Figure S-5: Time-course of functional Kv1.3 activity after stimulation with both anti-CD3 and anti-CD28 antibodies • Figure S-6: Distribution of Kv1.3 ion currents in regulatory T cells and dendritic cells
A voltage-gated potassium channel in human T lymphocytes
The Journal of Physiology, 1985
1. Human peripheral T lymphocytes were studied at 20-24 TC using the gigaohm seal recording technique in whole-cell or outside-out patch conformations. The predominant ion channel present under the conditions employed was a voltage-gated K+ channel closely resembling delayed rectifier K+ channels of nerve and muscle. 2. The maximum K+ conductance in ninety T lymphocytes ranged from 0-7 to 8-9 nS, with a mean of 4-2 nS. The estimated number of K+ channels per cell is 400, corresponding to a density of about three channels/sm2 apparent membrane area. 3. The activation of K+ currents could be fitted by Hodgkin-Huxley type n4 kinetics. The K+ conductance in Ringer solution was half-maximal at-40 mV. 4. The time constant of K+ current inactivation was practically independent of voltage except near the threshold for activating the K+ conductance. Recovery from inactivation was slow and followed complex kinetics. Steady-state inactivation was half-maximal at-70 mV, and was complete at positive potentials. 5. Permeability ratios, relative to K+, determined from reversal potential measurements were: K+(1-0) > Rb+(0-77) > NH4+(0-10) > Cs+(0-02) > Na+(< 0'01). 6. Currents through K+ channels display deviations from the independence principle. The limiting outward current increases when external K+ is increased, and Rb+ carries less inward current than expected from its relative permeability. 7. Tail current kinetics were slowed about 2-fold by raising the external K+ concentration from 4-5 to 160 mm, and were 5 times slower in Rb+ Ringer solution than in K+ Ringer solution. 8. Single K+ channel currents had two amplitudes corresponding to about 9 and 16 pS in Ringer solution. Replacing Ringer solution with isotonic K+ Ringer solution increased the unitary conductance and resulted in inward rectification of the unitary current-voltage relation. Comparable effects ofexternal K+ were seen in the whole-cell conductance and instantaneous current-voltage relation. 9. Several changes in the K+ conductance occurred during the first few minutes after achievement of the whole-cell conformation. Most are explainable by dissipation of a 10-20 mV junction potential between pipette solution and the cytoplasm, and by the use of a holding potential more negative than the resting potential. However, t Authors names are printed alphabetically. M. D. CAHALAN AND OTHERS inactivation of K+ currents became faster and more complete, changes not accounted for by these mechanisms. 10. K+ efflux through open K+ channels in intact lymphocytes, calculated from measured properties of K+ channels, can account for efflux values reported in resting lymphocytes, and for the increase in K+ efflux upon mitogenic stimulation. 11. Inward currents were observed in a few cells. These currents displayed voltage dependence, kinetics, unitary conductance, and pharmacological sensitivity characteristic of voltage-gated Na+ channel currents in excitable cells. Tetrodotoxin did not measurably inhibit phytohaemagglutinin-induced mitogenesis in T lymphocytes. 198 K+ CURRENTS IN HUMAN T L YMPHOCYTES dependent Ca2+ channels were never observed. The possible contribution of currents through voltage-gated K+ channels to reported K+ and Ca2+ fluxes in resting and mitogen-activated lymphocytes is discussed. Preliminary accounts ofthis work have appeared (DeCoursey et al. 1984 a; Cahalan, DeCoursey, Chandy & Gupta, 1984). METHODS Isolation of T lymphocytes. Heparinized (20 u./ml) peripheral venous blood was obtained from healthy volunteers. Lymphocytes from sixteen individuals were studied. Mononuclear cells were isolated by Ficoll-Hypaque (FH) density gradient, washed thrice with Ca2+-and Mg2+-free Hanks balanced salt solution (HBSS) supplemented with 25 mM-HEPES and resuspended in HBSS at 4 x 106 cells/ml. T lymphocytes were purified by resetting mononuclear cells with 2-aminoethyl isothiouronium bromide hydrobromide-treated sheep red blood cells, and separating rosetted T lymphocytes from non-rosetted non-T cells on FH gradient. Sheep red blood cells attached to T lymphocytes were lysed by Tris buffer (17 mM), pH 7-2, containing ammonium chloride (0-134 M). T lymphocytes were washed thrice with HBSS and resuspended in RPMI-1640 medium (Irvine Scientific Inc., Santa Ana, CA) containing 25 mM-HEPES, 10 % pooled heat-inactivated human AB serum, 100 u. penicillin/ml, 100 ,ug streptomycin/ml and 2 mM-L-glutamine (GIBCO, Grand Island, NY). The cells were > 95% viable determined by Trypan Blue dye exclusion and contained > 90% T lymphocytes as assessed by Leu 1 (Pan-T, Becton Dickinson, Mountainside, CA) monoclonal antibody binding and fluorescence-activated cell sorter/analyser (Becton-Dickinson, Sunnyvale, CA). Mitogenests studies. T lymphocytes were placed in microtitre wells in a total volume of 100 #s1, at 105/well, and incubated in the presence of 16 #sg PHA/ml (PHA-P, Difco, Detroit, MI) at 37°C in 5% CO2 for 60 h. The wells were pulsed with [3H]thymidine at 1 #sCi/well for the last 12 h. Gigaohm seal recording. Lymphocytes suspended in medium were placed in a small (300-400 #l) glass recording chamber and allowed to settle for a few minutes. The chamber was then flushed thoroughly with Ringer solution (Table 1). Pipette solutions containing as principal anion F-, Cl-, aspartateor glutamate-were tested (Table 1). T lymphocytes survived well, up to several hours, with F-, less well with aspartate-, and poorly with the other anions tested. For this reason KF was used in most experiments.
Ionic channels in murine macrophages
The Journal of Cell Biology, 1987
In this paper we examine the different voltage or calcium-dependent currents present in murine peritoneal macrophages, and in a macrophage-like cell line, J774. Three of these are K currents while the fourth is carried by CI. One K current, activated by hyperpolarization, has all the characteristics of the inward rectifier found in egg or muscle cells. It appears in peritoneal macrophages only after several days in culture. A second K current, activated by depolarization, is a typical delayed rectifier. The amplitude of these currents and, as a consequence, the membrane potential of the cells, can be markedly changed by the movement of fluid around the cells. A third K current is activated by internal calcium levels in the micromolar range. It presents a low-voltage sensitivity and is blocked by 0.1-1 mM quinine. The CI current flows through large-size channels (180-390 pS) that are active mainly in excised patches. These channels are unlikely to be half gap junctional channels, as suggested in former studies. The second goal of this paper is to examine if the activation of receptors for the Fc fragment of IgGs (Fc receptors) is associated with a change in the electrical properties of the membrane of macrophages. We have observed that the binding of multivalent ligands (the monoclonal antibody 2.4G2, aggregated IgGs, or sheep red blood cells coated with IgGs) to their Fc receptors on adherent macrophages did not trigger any change in resting potential. This is a surprising difference with former results obtained on non-adherent J774 cells (Young, J. D.-E., J. C. Unkeless, H.
Divalent ion trapping inside potassium channels of human T lymphocytes
The Journal of General Physiology, 1989
Using the patch-clamp whole-cell recording technique, we investigated the influence of external Ca 2+, Ba ~+, K +, Rb +, and internal Ca 2+ on the rate of K + channel inactivation in the human T lymphocyte-derived cell line, Jurkat E6-1. Raising external Ca 2+ or Ba ~+, or reducing external K +, accelerated the rate of the K + current decay during a depolarizing voltage pulse. External Ba ~+ also produced a use-dependent block of the K + channels by entering the open channel and becoming trapped inside. Raising internal Ca 2+ accelerated inactivation at lower concentrations than external Ca ~+, but increasing the Ca z+ buffering with BAPTA did not affect inactivation. Raising [K+]o or adding Rb + slowed inactivation by competing with divalent ions. External Rb + also produced a use-dependent removal of block of K + channels loaded with Ba 2+ or Ca 2+. From the removal of this block we found that under normal conditions ~25% of the channels were loaded with Ca ~+, whereas under conditions with 10 #M internal Ca ~+ the proportion of channels loaded with Ca ~+ increased to ~50%. Removing all the divalent cations from the external and internal solution resulted in the induction of a nonselective, voltage-independent conductance. We conclude that Ca 2+ ions from the outside or the inside can bind to a site at the K § channel and thereby block the channel or accelerate inactivation.
Ion channels and transporters in lymphocyte function and immunity
Nature Reviews Immunology, 2012
Lymphocyte function is regulated by a network of ion channels and transporters in the plasma membrane of B and T cells. These proteins modulate the cytoplasmic concentrations of diverse cations, such as calcium, magnesium and zinc ions, which function as second messengers to regulate crucial lymphocyte effector functions, including cytokine production, differentiation and cytotoxicity. The repertoire of ion-conducting proteins includes calcium release-activated calcium (CRAC) channels, P2X receptors, transient receptor potential (TRP) channels, potassium channels, chloride channels and magnesium and zinc transporters. This Review discusses the roles of ion conduction pathways in lymphocyte function and immunity.