High-throughput profiling of ion channel activity in primary human lymphocytes (original) (raw)
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Electroimmunology: the physiologic role of ion channels in the immune system
Journal of immunology (Baltimore, Md. : 1950), 1985
The recent development of the gigaohm seal voltage clamp technique has provided an approach to study individual cells of the immune system. Six distinct types of ion channels, most resembling channels found in nerve and muscle, have been identified in immune cells. Some of these channels appear to play important roles in various aspects of immune function. This article summarizes recent studies on ion channels in lymphocytes and macrophages.
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.
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.
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 ...
Voltage-gated potassium channels are required for human T lymphocyte activation
Journal of Experimental Medicine, 1984
The calcium channel blockers, verapamil and diltiazem, inhibit phytohemagglutinin (PHA)-induced mitogenesis at concentrations that block the T lymphocyte K channel currents. K channel blockers also inhibit the allogeneic mixed lymphocyte response in a dose-dependent manner with the same potency sequence as for block of K currents. K channel blockers inhibit PHA-stimulated mitogenesis only if added during the first 20-30 h after PHA addition, but not later, indicating a requirement for functional K channels during this period. We investigated the effect of K channel blockers on various aspects of protein synthesis for two reasons: first, protein synthesis appears to be necessary for the events leading to DNA synthesis, and second, the increase in the protein synthetic rate commences during the first 24-48 h after PHA addition. PHA-induced total protein synthesis was reduced to the level in unstimulated T lymphocytes by K channel blockers in a dose-dependent manner with the same poten...
Proceedings of the National Academy of Sciences, 2003
Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3͞FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3͞FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C ؍ 0.64). A different hierarchical level of molecular proximity between Kv1.3͞FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E ؍ 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.
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.
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 ...
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.