Electroimmunology: the physiologic role of ion channels in the immune system (original) (raw)
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.
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.
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.
Mitogen induction of ion channels in murine T lymphocytes
Journal of General Physiology, 1987
Using gigohm-seal recording, we studied ion channel expression in resting and activated T lymphocytes from mice. Both the number of channels per cell and the predominant type of K+ channel depend upon the state of activation of the cell. Unstimulated T cells express small numbers of K+ channels, typically a dozen per cell, and are heterogeneous, usually expressing either type n or type l K+ channels (see DeCoursey, T. E., K. G. Chandy, S. Gupta, and M. D. Cahalan. 1987. Journal of General Physiology. 89:379-404). 1 d after stimulation by the murine T cell mitogen concanavalin A, large numbers of type n K+ channels appear in enlarged, activated cells. Type n channels appear in activated cells with a time course consistent with that reported for mitogen-induced enhancement of protein synthesis. Voltage-gated tetrodotoxin-sensitive Na+ channels present in about one-third of unstimulated cells from the MRL-n strain are increased approximately 10-fold after activation.
Modulation of Adaptive Immunity and Viral Infections by Ion Channels
Frontiers in Physiology, 2021
Most cellular functions require of ion homeostasis and ion movement. Among others, ion channels play a crucial role in controlling the homeostasis of anions and cations concentration between the extracellular and intracellular compartments. Calcium (Ca2+) is one of the most relevant ions involved in regulating critical functions of immune cells, allowing the appropriate development of immune cell responses against pathogens and tumor cells. Due to the importance of Ca2+ in inducing the immune response, some viruses have evolved mechanisms to modulate intracellular Ca2+ concentrations and the mobilization of this cation through Ca2+ channels to increase their infectivity and to evade the immune system using different mechanisms. For instance, some viral infections require the influx of Ca2+ through ionic channels as a first step to enter the cell, as well as their replication and budding. Moreover, through the expression of viral proteins on the surface of infected cells, Ca2+ channe...
Selectivity and gating of the type L potassium channel in mouse lymphocytes
The Journal of General Physiology, 1991
Type l voltage-gated K+ channels in murine lymphocytes were studied under voltage clamp in cell-attached patches and in the whole-cell configuration. The kinetics of activation of whole-cell currents during depolarizing pulses could be fit by a single exponential after an initial delay. Deactivation upon repolarization of both macroscopic and microscopic currents was mono-exponential, except in Rb-Ringer or Cs-Ringer solution in which tail currents often displayed "hooks," wherein the current first increased or remained constant before decaying. In some cells type l currents were contaminated by a small component due to type n K+ channels, which deactivate approximately 10 times slower than type l channels. Both macroscopic and single channel currents could be dissected either kinetically or pharmacologically into these two K+ channel types. The ionic selectivity and conductance of type l channels were studied by varying the internal and external permeant ion. With 160 mM ...
Cellular Immunology, 1987
The in vitro effect of ion channel-blocking agents verapamil (V), 4-aminopyridine (4AP), tetraethylammonium (TEA), and quinine (Q) was examined on the proliferative response of human peripheral blood T lymphocytes in the autologous mixed-lymphocyte reaction (AMLR). All the above channel blockers in a dose-dependent manner inhibited the AMLR. Tetramethylammonium (TMA), an analog of TEA that does not block K' channel currents, did not inhibit the AMLR. 4AP at 1 W/ml concentration inhibited the expression of IL-2 receptors, as defined by monoclonal antibody anti-Tat, on T-cell activated in the AMLR. In vitro addition of recombinant interleukin 2 (rIL2) completely corrected the inhibition of the AMLR by channel blockers. Furthermore, the concentrations of ion channel blockers required for blocking 50% response of T cells in the AMLR was much lower than that reported for 50% block of T-cell proliferation in response to phytohemagglutinin or in allogeneic mixed-lymphocyte culture (MLC). These data suggest a role of ion channels in T-cell functions and show that the AMLR provides a more sensitive system, as compared to lectin stimulation or MLC, to examine any immunosuppressive effects of ion channel-blocking agents in disease states where they are used as therapeutic modalities. 0 1987 Academic Press, Inc.
III. Ion Channel Expression in PMA-differentiated Human THP-1 Macrophages
Journal of Membrane Biology, 1996
Ion channel expression was studied in THP-1 human monocytic leukemia cells induced to differentiate into macrophage-like cells by exposure to the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Inactivating delayed rectifier K + currents, I DR , present in almost all undifferentiated THP-1 monocytes, were absent from PMA-differentiated macrophages. Two K + channels were observed in THP-1 cells only after differentiation into macrophages, an inwardly rectifying K + channel (I IR) and a Ca 2+-activated maxi-K channel (I BK). I IR was a classical inward rectifier, conducting large inward currents negative to E K and very small outward currents. I IR was blocked in a voltage-dependent manner by Cs + , Na + , and Ba 2+ , block increasing with hyperpolarization. Block by Na + and Ba 2+ was time-dependent, whereas Cs + block was too fast to resolve. Rb + was sparingly permeant. In cell-attached patches with high [K + ] in the pipette, the single I IR channel conductance was ∼30 pS and no outward current could be detected. I BK channels were observed in cell-attached or inside-out patches and in whole-cell configuration. In cell-attached patches the conductance was ∼200-250 pS and at potentials positive to ∼100 mV a negative slope conductance of the unitary current was observed, suggesting block by intracellular Na +. I BK was activated at large positive potentials in cell-attached patches; in inside-out patches the voltageactivation relationship was shifted to more negative potentials by increased [Ca 2+ ]. Macroscopic I BK was blocked by external TEA + with half block at 0.35 mM. THP-1 cells were found to contain mRNA for Kv1.3 and IRK1. Levels of mRNA coding for these K + channels were studied by competitive PCR (polymerase chain reaction), and were found to change upon differentiation in the same direction as did channel expression: IRK1 mRNA increased at least 5-fold, and Kv1.3 mRNA decreased on average 7-fold. Possible functional correlates of the changes in ion channel expression during differentiation of THP-1 cells are discussed.
Journal of General Physiology, 1993
Ca(2+)-activated K+[K(Ca)] channels in resting and activated human peripheral blood T lymphocytes were characterized using simultaneous patch-clamp recording and fura-2 monitoring of cytosolic Ca2+ concentration, [Ca2+]i. Whole-cell experiments, using EGTA-buffered pipette solutions to raise [Ca2+]i to 1 microM, revealed a 25-fold increase in the number of conducting K(Ca) channels per cell, from an average of 20 in resting T cells to > 500 channels per cell in T cell blasts after mitogenic activation. The opening of K(Ca) channels in both whole-cell and inside-out patch experiments was highly sensitive to [Ca2+]i (Hill coefficient of 4, with a midpoint of approximately 300 nM). At optimal [Ca2+]i, the open probability of a K(Ca) channel was 0.3-0.5. K(Ca) channels showed little or no voltage dependence from -100 to 0 mV. Single-channel I-V curves were linear with a unitary conductance of 11 pS in normal Ringer and exhibited modest inward rectification with a unitary conductance ...
Increase in voltage gated potassium currents of human lymphocytes on culture
Indian journal of experimental biology, 2012
Voltage gated potassium channels present in T lymphocytes play an important role during lymphocyte activation. Though an increase in potassium currents has been reported in activated lymphocytes, changes in potassium currents in culture without activation by antigen or mitogen has not been reported. The peak potassium current densities on day 1 and day 5 of culture have been compared in this study. Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation. Lymphocytes were separated from PBMCs by negative selection using anti-CD14 coated magnetic beads and cultured under appropriate conditions without antigenic or mitogenic stimulation. Lymphocytes were patched on day 1 or day 5 of culture. Voltage gated potassium currents were recorded by whole cell patch clamp technique using a depolarizing protocol. The mean of peak current densities recorded at +60 mV on day 1 of culture was 228.12 +/- 89.39 pA/pF (n = 7) and on day 5 of culture was 468.96 +/- ...