Two types of potassium channels in murine T lymphocytes

The properties of two types of K+ channels in murine T lymphocytes are described on the basis of whole-cell and isolated-patch recordings using the gigohm-seal technique. Type l (standing for "lpr gene locus" or "large") channels were characterized mainly in T cells from mutant MRL/MpJ-lpr/lpr mice, in which they are present in large numbers. Type n ("normal") K+ channels are abundant and therefore most readily studied in concanavalin A-activated T cells from four strains of mice, MRL-+/+, CBA/J, C57BL/6J, and BALB/c. Type l channels, compared with type n, are activated at potentials approximately 30 mV more positive, and close much more rapidly upon repolarization. Type l channels inactivate more slowly and less completely than type n during maintained depolarization, but recover from inactivation more rapidly, so that little inactivation accumulates during repetitive pulses. Type l channels have a higher unitary conductance (21 pS) than type n (12 pS) and are less sensitive to block by external Co++, but are 100-fold more sensitive to block by external tetraethylammonium (TEA), with half-block of type l channels at 50-100 microM TEA compared with 8-16 mM for type n. TEA blocks both types of channels by reducing the apparent single channel current amplitude, with a dose-response relation similar to that for blocking macroscopic currents. Murine type n K+ channels resemble K+ channels in human T cells.     

Charybdotoxin blocks voltage-gated K+ channels in human and murine T lymphocytes

A variety of scorpion venoms and purified toxins were tested for effects on ion channels in human T lymphocytes, a human T leukemia cell line (Jurkat), and murine thymocytes, using the whole-cell patch-clamp method. Nanomolar concentrations of charbdotoxin (CTX), a purified peptide component of Leiurus quinquestriatus venom known to block Ca2+-activated K+ channels from muscle, blocked "type n" voltage-gated K+ channels in human T lymphoid cells. The Na+ channels occasionally expressed in these cells were unaffected by the toxin. From the time course of development and removal of K+ channel block we determined the rates of CTX binding and unbinding. CTX blocks K+ channels in Jurkat cells with a Kd value between 0.5 and 1.5 nM. Of the three types of voltage-gated K+ channels present in murine thymocytes, types n and n' are blocked by CTX at nanomolar concentrations. The third variety of K+ channels, "type l," is unaffected by CTX. Noxiustoxin (NTX), a purified toxin from Centruroides noxius known to block Ca2+-activated K+ channels, also blocked type n K+ channels with a high degree of potency (Kd = 0.2 nM). In addition, several types of crude scorpion venoms from the genera Androctonus, Buthus, Centruroides, and Pandinus blocked type n channels. We conclude that CTX and NTX are not specific for Ca2+ activated K+ channels and that purified scorpion toxins will provide useful probes of voltage-gated K+ channels in T lymphocytes. The existence of high-affinity sites for scorpion toxin binding may help to classify structurally related K+ channels and provide a useful tool for their biochemical purification.     

Ca(2+)-activated K+ channels in human leukemic T cells

Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.     

CD4-CD8- T cells from mice with collagen arthritis display aberrant expression of type l K+ channels

Expression of voltage-gated K+ channels in mAb-defined T cell subsets from normal mice and mice with experimental autoimmune arthritis was studied with the patch-clamp whole-cell recording technique in combination with fluorescence microscopy. CD4+CD8- Th cells from DBA/1 LacJ mice with type II collagen arthritis expressed low levels of type n K+ channels, and CD4-CD8+ T cells (cytotoxic) showed small numbers of type l or n' K+ channels, like their phenotypic counterparts in normal mice. CD4-CD8-Thy-1.2+ (double negative or DN) T cells from the diseased mice, however, displayed an abundance of type l K+ channels compared to DN T cells in normal mice, or mice immunized with CFA. Furthermore, the aberrant expression of type l K+ channels correlated with the presence of active disease. DN T cells from mice with SLE, type-1 diabetes mellitus, and experimental allergic encephalomyelitis, also exhibited a high number of type l K+ channels. These results suggest that expression of numerous type l K+ channels may be a useful marker for DN T cells associated with these autoimmune disorders.     

Calcium-activated potassium channels in resting and activated human T lymphocytes. Expression levels, calcium dependence, ion selectivity, and pharmacology

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 of approximately 35 pS in symmetrical 160 mM K+. Permeability ratios, relative to K+, determined from reversal potential measurements were: K+ (1.0) > Rb+ (0.96) > NH4+ (0.17) > Cs+ (0.07). Slope conductance ratios were: NH4+ (1.2) > K+ (1.0) > Rb+ (0.6) > Cs+ (0.10). Extracellular Cs+ or Ba2+ each induced voltage-dependent block of K(Ca) channels, with block increasing at hyperpolarizing potentials in a manner suggesting a site of block 75% across the membrane field from the outside. K(Ca) channels were blocked by tetraethylammonium (TEA) applied externally (Kd = 40 mM), but were unaffected by 10 mM TEA applied inside by pipette perfusion. K(Ca) channels were blocked by charybdotoxin (CTX) with a half-blocking dose of 3-4 nM, but were resistant to block by noxiustoxin (NTX) at 1-100 nM. Unlike K(Ca) channels in Jurkat T cells, the K(Ca) channels of normal resting or activated T cells were not blocked by apamin. We conclude that while K(Ca) and voltage-gated K+ channels in the same cells share similarities in ion permeation, Cs+ and Ba2+ block, and sensitivity to CTX, the underlying proteins differ in structural characteristics that determine channel gating and block by NTX and TEA.     

Abundant expression of type l K+ channels. A marker for lymphoproliferative diseases?

Using the patch clamp whole-cell recording technique, we studied expression of K+ channels in mAb-defined T cell subsets from diseased C3H-lpr/lpr and C3H-gld/gld mice and from healthy C3H-HeJ congenic controls. Both mutant mouse strains develop a lupus-like syndrome accompanied by hyperplasia of a functionally and phenotypically abnormal T cell subset. These defective cells, which are Thy-1.2+ CD4- CD8- B220+ F23.1+, display an abundance of type l K+ channels. Phenotypically similar lymph node T cells from normal C3H-HeJ mice, or young C3H-lpr/lpr mice before the onset of disease, do not display large numbers of type l K+ channels. CD4+ CD8- T cells (helper/inducer) from the mutant mice express a small number of type n K+ channels, and CD4- CD8+ T cells (suppressor/cytotoxic) show a low level of type l or n' K+ channels, as do their phenotypically equivalent counterparts in the normal mouse thymus. These results suggest that the abundant expression of type l K+ channels is a marker for the defective lpr and gld T cell subset and may reflect the "abnormal" proliferative status of these cells.     

Two K+ channel types, muscarinic agonist-activated and inwardly rectifying, in a Cl- secretory epithelium: the avian salt gland

Patches of membrane on cells isolated from the nasal salt gland of the domestic duck typically contained two types of K+ channel. One was a large-conductance ("maxi") K+ channel which was activated by intracellular calcium and/or depolarizing membrane voltages, and the other was a smaller-conductance K+ channel which exhibited at least two conductance levels and displayed pronounced inward rectification. Barium blocked both channels, but tetraethylammonium chloride and quinidine selectively blocked the larger K+ channel. The large K+ channel did not appear to open under resting conditions but could be activated by application of the muscarinic agonist, carbachol. The smaller channels were open under resting conditions but the gating was not affected by carbachol. Both of these channels reside in the basolateral membranes of the Cl- secretory cells but they appear to play different roles in the life of the cell.     

T淋巴细胞上的离子通道

近年的研究证明,淋巴细胞上的离子通道,在免疫功能调节中具有重要的作用。T淋巴细胞上主要有三类离子通道,即Ca2 、K 和C1-通道。Ca2 通过T淋巴细胞膜上的Ca2 通道(CRAC)进入细胞内,可作为第二信使激活T淋巴细胞。通过K 通道的K 外流是T淋巴细胞膜电位形成的基础。由于膜电位水平可以影响钙离子的内流,因此,K 通道可以间接调节T淋巴细胞的活化和功能。T淋巴细胞上的Cl-通道是新近发现的一种离子通道,可能与细胞的体积调节有关。本文扼要总结了T淋巴细胞上离子通道的新近进展。     

The antidiabetic sulfonylurea glibenclamide is a potent blocker of the ATP-modulated K+ channel in insulin secreting cells

The ATP-sensitive K+ channel of RINm5F insulinoma cells is activated after an intracellular ATP depletion. This activation can be followed by 86Rb+ efflux. Once activated by ATP depletion, the K+ channel can be blocked by the hypoglycemic drug, glibenclamide. The blockade is of a high-affinity type (K0.5 = 0.06 nM). Recording of the activity of ATP-sensitive K+ channels with the patch-clamp technique confirmed that they could be completely blocked with 20 nM glibenclamide.     

Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells

Although the crucial role of Ca(2+) influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca(2+) channels in normal human T lymphocytes. The use of Na(+) as the permeant ion in divalent-free solution permitted Ca(2+) release-activated Ca(2+) (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions. The number of CRAC channels per cell increased greatly from approximately 15 in resting T cells to approximately 140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to approximately 60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 microM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling.     

Intracellular signalling involved in activation of the volume-sensitive K+ current in Ehrlich ascites tumour cells

The cell swelling-activated K+ channel in Ehrlich ascites tumour cells has a conductance of 5 pS estimated from noise analysis of the volume-sensitive whole-cell K+ current (I(K,vol)). I(K,vol) exhibits Goldman-Hodgkin-Katz type behaviour and is insensitive to clotrimazole, apamin and charybdotoxin (ChTX), but inhibited by clofilium. Its small conductance, lack of intrinsic voltage-dependence and peculiar pharmacological profile are similar to properties described for the two-pore domain background K+ TASK channels. Neither Ca2+ nor ATP work as initiators in the activation of I(K,vol). In contrast, several investigations in Ehrlich cells suggest an important role for leukotriene D4 (LTD4) in the activation of I(K,vol). Under isotonic conditions, LTD4 activates Ca2+-dependent, ChTX-sensitive K+ channels as well as Ca2+-independent. ChTX-insensitive K+ channels. The LTD4-activated, ChTX-insensitive K+ current exhibits a current-voltage relation, pharmacological profile and single channel conductance similar to that of I(K,vol), indicating that LTD4 is the signalling molecule responsible for activation of the volume-sensitive K+ channels in Ehrlich cells. Hypotonic swelling of Ehrlich cells results in translocation of the 85-kDa cytosolic (c) PLA2alpha to the nucleus where it is activated. This activation leads to an increase in arachidonic acid release followed by an increased release of leukotrienes, and is essential in cell swelling-induced activation of I(K,vol) and of the organic osmolyte channels.     

Role of TASK2 in the control of apoptotic volume decrease in proximal kidney cells

Apoptotic volume decrease (AVD) is prerequisite to apoptotic events that lead to cell death. In a previous study, we demonstrated in kidney proximal cells that the TASK2 channel was involved in the K+ efflux that occurred during regulatory volume decrease. The aim of the present study was to determine the role of the TASK2 channel in the regulation of AVD and apoptosis phenomenon. For this purpose renal cells were immortalized from primary cultures of proximal convoluted tubules (PCT) from wild type and TASK2 knock-out mice (task2-/-). Apoptosis was induced by staurosporine, cyclosporin A, or tumor necrosis factor alpha. Cell volume, K+ conductance, caspase-3, and intracellular reactive oxygen species (ROS) levels were monitored during AVD. In wild type PCT cells the K+ conductance activated during AVD exhibited characteristics of TASK2 currents. In task2-/- PCT cells, AVD and caspase activation were reduced by 59%. Whole cell recordings indicated that large conductance calcium-activated K+ currents inhibited by iberiotoxin (BK channels) partially compensated for the deletion of TASK2 K+ currents in the task2-/- PCT cells. This result explained the residual AVD measured in these cells. In both cell lines, apoptosis was mediated via intracellular ROS increase. Moreover AVD, K+ conductances, and caspase-3 were strongly impaired by ROS scavenger N-acetylcysteine. In conclusion, the main K+ channels involved in staurosporine, cyclosporin A, and tumor necrosis factor-alpha-induced AVD are TASK2 K+ channels in proximal wild type cells and iberiotoxin-sensitive BK channels in proximal task2-/- cells. Both K+ channels could be activated by ROS production.     

BKCa channels activating at resting potential without calcium in LNCaP prostate cancer cells

Large-conductance Ca2+-dependent K+ (BK(Ca)) channels are activated by intracellular Ca2+ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK(Ca)-type K+ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK(L). K+ selectivity, high conductance, activation by Mg2+ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK(Ca) channels. However, unlike conventional BK(Ca) channels, BK(L) channels activated in the absence of free cytosolic Ca2+ at physiological membrane potentials; the half-maximal activation voltage was shifted by about -100 mV compared with BK(Ca) channels. Half-maximal Ca2+-dependent activation was observed at 0.4 microM: for BK(L) (at -20 mV) and at 4.1 microM: for BK(Ca) channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK(L) conductance. Expression of hSlo-beta1 in LNCaP cells shifted voltage-dependent activation to values between that of BK(L) and BK(Ca) channels and reduced the slope of the P (open) (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK(Ca) subunit, which is responsible for the BK(L) phenotype in a dominant manner. BK(L)-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK(L) in LNCaP cells is regulated by serum-derived factors, however not by androgens.     

Regulation of a calcium-sensitive K+ channel (cIK1) by protein kinase C

Ca2+-sensitive K+ channels (IK1 channels) are required for many physiological functions such as cell proliferation, epithelial transport or cell migration. They are regulated by the intracellular Ca2+ concentration and by phosphorylation-dependent reactions. Here, we investigate by means of the patch-clamp technique mechanisms by which protein kinase C (PKC) regulates the canine isoform, cIK1, cloned from transformed renal epithelial (MDCK-F) cells. cIK1 elicits a K+-selective, inwardly rectifying, and Ca2+-dependent current when expressed in HEK293 or CHO cells. It is inhibited by charybdotoxin, clotrimazole, and activated by 1-ethyl-2-benzimidazolone. cIK1 is activated by intracellular application of ATP or ATP[gS]. ATP-dependent activation is reversed by PKC inhibitors (bisindolylmaleimide, calphostin C), while stimulation with ATP[gS] resists PKC inhibition. Stimulation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) leads to the acute activation of cIK1 currents, which are blocked by PKC inhibitors. In contrast, PKC depletion by overnight incubation with PMA prevents ATP-dependent cIK1 activation. Neither single mutations nor the simultaneous mutation of all PKC sites (T101, S178, T329) to alanine alter the acute regulation of cIK1 channels by PKC. However, current amplitudes of CIK1-T329A and the triple mutant are dramatically increased upon long-term treatment with PMA. These mutations thereby disclose an inhibitory effect on cIKl current of the PKC site at T329. Our results indicate that cIK1 channel activity is regulated in two ways. PKC-dependent activation of cIK1 channels occurs indirectly, while the inhibitory effect probably requires a direct interaction with the channel protein.     

Development of ionic channels during mouse neuronal differentiation

Using a mouse embryonal teratocarcinoma (E.C.) cell line, it was possible to follow the sequence of development of ionic channels during neuronal differentiation, with patch-clamp techniques. 1003 E.C. cells were induced to differentiate into neurons by culturing them in defined medium without foetal calf serum (DARMON et al., 1981). Non-differentiated cells were not excitable and presented mainly 2 types of K+ channels: a Ca2+ activated K+ channel (220 pS in symmetrical K+) and a delayed rectifier (30 pS in symmetrical K+). When the cells start to grow neurites, a low threshold calcium current can be recorded, only if the cell is held at hyperpolarized potentials (-70 to -80 mV). Fully differentiated cells with long neurites presented a complete repertoire of ionic channels: voltage dependent Na+ and Ca2+ channels, Ca2+ activated K+ channel and K+ delayed rectifier.     

Voltage-gated and Ca(2+)-activated K+ channels in intact human T lymphocytes. Noninvasive measurements of membrane currents, membrane potential, and intracellular calcium

Voltage-gated n-type K(V) and Ca(2+)-activated K+ [K(Ca)] channels were studied in cell-attached patches of activated human T lymphocytes. The single-channel conductance of the K(V) channel near the resting membrane potential (Vm) was 10 pS with low K+ solution in the pipette, and 33 pS with high K+ solution in the pipette. With high K+ pipette solution, the channel showed inward rectification at positive potentials. K(V) channels in cell-attached patches of T lymphocytes inactivated more slowly than K(V) channels in the whole-cell configuration. In intact cells, steady state inactivation at the resting membrane potential was incomplete, and the threshold for activation was close to Vm. This indicates that the K(V) channel is active in the physiological Vm range. An accurate, quantitative measure for Vm was obtained from the reversal potential of the K(V) current evoked by ramp stimulation in cell-attached patches, with high K+ solution in the pipette. This method yielded an average resting Vm for activated human T lymphocytes of -59 mV. Fluctuations in Vm were detected from changes in the reversal potential. Ionomycin activates K(Ca) channels and hyperpolarizes Vm to the Nernst potential for K+. Elevating intracellular Ca2+ concentration ([Ca2+]i) by ionomycin opened a 33-50-pS channel, identified kinetically as the CTX-sensitive IK-type K(Ca) channel. The Ca2+ sensitivity of the K(Ca) channel in intact cells was determined by measuring [Ca2+]i and the activity of single K(Ca) channels simultaneously. The threshold for activation was between 100 and 200 nM; half-maximal activation occurred at 450 nM. At concentrations > 1 microM, channel activity decreased. Stimulation of the T-cell receptor/CD3 complex using the mitogenic lectin, PHA, increased [Ca2+]i, and increased channel activity and current amplitude resulting from membrane hyperpolarization.     

Charybdotoxin-sensitive, Ca(2+)-dependent membrane potential changes are not involved in human T or B cell activation and proliferation.

The involvement of ion channels in B and T lymphocyte activation is supported by many reports of changes in ion fluxes and membrane potential after mitogen binding. Human T and B lymphocytes demonstrate an early and transient hyperpolarization after ligand binding. Inasmuch as the change in membrane potential is dependent on elevation of free cytosolic calcium, the hyperpolarization is presumably through opening of Ca(2+)-stimulated K+ channels. We have used charybdotoxin, a known inhibitor of Ca(2+)-dependent K+ channels, to study the role of these channels in lymphocyte activation and mitogenesis. We demonstrate that charybdotoxin inhibits the ligand-induced transient membrane hyperpolarization in B and T cells in a dose-dependent fashion, without affecting changes in cytosolic Ca2+. However, blockade of the Ca(2+)-activated K+ channel is not associated with changes in cell-cycle gene activation, IL-2 production, IL-2R expression or B and T cell mitogenesis. These results imply that membrane potential changes secondary to the ligand-dependent opening of Ca(2+)-activated K+ channels are not involved in B and T lymphocyte activation and mitogenesis.