electroimmunology: the physiologic role of ion channels in ... - CiteSeerX

0022-1767/85/1352-787S$02.00/0 THEJOURNAL OF lUMUN0LoCY Copyright 0 1985 by The Amerlcan Association of Immunologlsts

Vol. 135. No. 2. August 1985. Prlnted In U.S.A.

ELECTROIMMUNOLOGY: THE PHYSIOLOGICROLE IMMUNE SYSTEM’

OF ION CHANNELS IN THE

K. GEORGE CHANDY,’ THOMAS E. DECOURSEY,” MICHAEL D. CAHALAN,”

AND

SUDHIR GUPTA

From the Departmentsof Medicine and ‘Physiology and Biophysics, University of California, Irvine. CA 9271 7

open “ligand-gated’’channels such as the acetylcholine receptor channel, or it may modulate channel function indirectly via a n intracellular second messenger, a s in the case of cardiac P-receptor-modulated Ca2+channels. Many channels aremodulated by more than one of these mechanisms, a s in the case of Ca2+-activated K+ channels, whose voltage dependence is a function of the cytoplasmic free Ca2+concentration. Several dozen distinct types of ion channels are known,characterized by their selective permeability to specific ions, by their voltage dependence, conductance, and gating kinetics, and by their sensitivity to specific drugs and toxins (reviewed in The immune and neuroendocrine systems receive and respond to a n almost unlimited array of diverse stimuli Reference 1). Ion channels have been studied extensively in excitable and retain a memory of the interaction. A complicated tissues. In part, this has been so because they clearly network of cell-cell interactions is utilized to achievethis degree of versatility. It is well known that ion channels play a n important role in these cells, but, in part, the are essential to the function of excitable cells, nerve and choice of preparation has been dictated by experimental muscle. This paper summarizes recent studies which necessity. The squid giant axon, with a diameter of 500 pm or more allows wire electrodes. along with capillary describe several types of ion channels present in the membranes of cells of the immune system. While their tubes for internal perfusion, tobe inserted longitudinally precise physiologic function has not yet been completely for voltage clamp recording. Conventional electrophysioworked out, there is growing evidence that ion channels logic techniques, in particular the intracellular microplay a n essential role in various aspects of cell function. electrode, are useful only in moderately large cells. InserIn this brief review, we focus primarily upon T lympho- tion of a conventional microelectrode into anycell results cytes and the role of a potassium ion-selective channel in a n unavoidable electricalleak around theelectrode. In in activation by mitogens. Ion channels present in B a large cell with thousands or millions of ion channels in may be insignificant, but lymphocytes, natural killer cells, and macrophages are its membrane, this leak current in a small cell such a s a lymphocyte, the electrode leak also discussed. Ion channels. Ions traverse cell membranes ina variety current may be much larger than that flowing through membrane. The gigaohm seal recordof ways. their distribution across the membrane being ion channels in the responsible for the membrane potential. Ions may be ing technique (reviewed in Reference 2) has revolutionit possible to record transported passively down their electrochemical gra- ized research on small cells, making dient throughion channels or by ion carriers or exchange faithfully currents as small a s those flowing through ammechanisms. Ions may also be actively transported single ion channels, a few pA (picoamperes = peres) or less. This capability presents the unique oppor“uphill” by energy-dependent pumps such as Na-K ATPase. In this paper we deal exclusively with ion channels, tunity to study the behavior of a single protein moiety, which are capableof transporting large numbers of spe- a n individual ion channel, in a small cell. In the “patchcific ions across cell membranes, a process whichcan be clamp” technique a firepolished glass micropipette with controlled by the cell on a very brief time scale. Ions a tip diameter of about 1 pm is filled with a solution of channels are pores formed by integral membrane pro- known ion composition. An electrode, usually achlorided teins. These pores may be open (conducting) or closed silver wire housed inside the pipette, measures the cur(nonconducting). Transitions between open and closed rent between the pipette and a bath electrode. The pipette states are termed “gating.” Gating can be regulated in is pressed against the surface of a cell and then gentle differentways.The probability that a “voltage-gated suction is applied, which, under favorable conditions, channel will open depends in a predictable manner di- results in formation of a tight electrical seal(witha rectly upon the cell membrane potential. Binding of ex- resistance of several gigaohms; (giga = 10’) between the “cell-attached patch” tracellular ligand to specific cell receptors may directly pipette and the membrane. This conformation enables measurement of discrete steps of current corresponding to opening and closing of single ‘This work was supported by National Institutes of Health Grants NS14609, AI-20717, and AG-04361 anda fellowship to K.G.C. from the ion channels in the patch.A large pulse of suction applied Southern California Affiliate, American Diabetic Association. to the pipette interior can be used to rupture the memTo whom correspondence should be addressed,Department of Medicine, Universityof California. Irvine. CA 92717. brane patch at the tip of the pipette, resulting in conti-

The recent developmentof the gigaohm Seal voltage clamp technique has provided an approach to study individual cells of the immune system. six distincttypes ofion channels, mostresembling channels foundinnerveand muscle,havebeen identified in immunecells. Some of these channels appear to play importantroles in variousaspects of immune function. This article summarizes recent studies on ion channels in lymphocytes and macrophages.

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ROLE OF ION CHANNELS IN CELLS IMMUNE

nuity of the cytoplasm and the pipette solution-the tion,increased K+ fluxes, and elevated cystosolic-free “whole cell conformation.” The physical stability of the Ca’+ that accompany T cell activation, based upon a pipette glass-to-membrane seal minimizes the leakage single type of channel-a voltage-gated K+ channel. current under the pipette. The pipette solution rapidly About 300 to 500 voltage-gated K+ channels are exequilibrates with the cytoplasm permitting control of in- pressed in the average human T cell. The kinetic and tracellular ion concentrations. Electrically, the cell is pharmacologic properties of T cell K+channels are very voltage-clamped to the potential within the pipette, and similar to those of “delayedrectifier” K+channels in nerve thus themembrane potential can be experimentally con- and muscle cells (3, 5).The voltage dependence of these trolled while ion currents passingthrough the entirecell channels and maximum K+ conductance are indistinmembrane are recorded. Ion channels in the cell mem- guishable with freeCa2+levels in pipette solutions rangbrane canthereby be identified and characterized on the ing from 2 nM to 1 FM. Since the pipette solution equilibasis of their voltage dependence, kinetics, and phar- brates rapidly with the cytoplasm, this K+ channel apmacologic properties. pears insensitive to freeCa+ concentrations well beyond Ion channels control a variety of cellular processes. the physiologic range (3,5). They are responsible for action potentials, transmitter The T cell mitogens phytohemagglutinin (PHA) and release, and postsynaptic responses, and are thusabso- concanavalin A (Con A) cause K+ channels to open more lutely essential for muscle contraction, the heart beat rapidly and atmore negative voltages in voltage-clamped and all neural activity. They alsoplay a role in the secre- T cells (3, 5, unpublished data). In a n intact T cell mitotion of hormones. Abnormalitiesof ion channels contrib- gens would increase theprobability of K+ channel openute to the pathogenesis of myotonia congenita and myas- ing at the resting potential, resulting in membrane hythenia gravis. Major functions of immune cells include perpolarization and increased K+ fluxes. Based upon a chemotaxis, cell growth and proliferation, production and kinetic model of K+ channel gating, we estimate that at secretion of antibodies and lymphokines, and killing of the normal T cell resting membrane potential of -50 to foreign or virus-infected cells. We and severalother -70 mV (see Reference 5), 0.1 to2 K+ channels would be workers have studiedthe involvement of ion channels in open at a given time. The estimated K+ efflux through several of these processes. Thus far, five types of chan- these open channels would be 10 to 400 attomoles (atto nels resembling ion channels found in excitable cells = 10”*)per min per cell, a range encompassing measured have been reported in various cells of the immune sys- passive K+ efflux values in resting human lymphocytes tem: a voltage-gated K+ channel with properties similar (see Reference 5). Mitogens would approximately double to those of the delayed rectifier in nerve ormuscle (3-7), the number of open channels, accounting for the rapid a voltage-gated Na+ channel (5)that seemsindistinguish- twofold increase in K+ flux that has been reported (see able from that found in nerve or muscle, a voltage-gated Reference 5). One day after mitogen activation, the K+ Ca2+channel with many similarities toCa2+channels in conductancein human T lymphocytes approximately nerves (8, 9). a Ca’+-activated K+ channel (lo),and an doubles (4). Because activated T cells are larger than inwardly rectifying K+ channel (7) that turns on when resting T cells, this result suggests that the density of may be maintainedafter the membraneis hyperpolarized. Another type of channel channels in the membrane is formed by macrophage Fc receptors in bilayers (1 1). activation. K+ channel expression is strikingly different inhuman Here we summarize recent studiesin which electrophysiologic techniques have been combined with standard and mouse T cells. While quiescent human T cells eximmunologic and biochemical methods in order toeluci- press several hundred K+ channels (3, 5),resting mouse date the role of ion channels in various aspects of im- T cells express a much smaller number of K+ channels (16,unpublished data). Preliminary studies indicatethat mune function. T lymphocyte activation. Severallines of evidence mitogen activation of mouse T cells after 1 day results in suggest that two signals arerequired for T cell activation: a substantial increase (roughly 10-fold) in the numbers one Ca2+dependent and the other macrophage orphorbol of conducting K+ channels per cell (16).In human T cells, ester dependent (12). These two signals initiate a series increases in K+ fluxes occur almost immediately after of events that culminatein cell division. Several workers mitogen addition, while in mouse T cells, elevated in K+ have postulated the existence of Ca2+channels andCa2+- fluxes develop after a delay of 6 to 8 hr ( 1 7). Thedelay in activated K+channels to account for the Ca2+ influx and mitogen-enhanced K+ fluxes in mouse T cells may be the accompanying membrane hyperpolarization and in- related to the delay in the expression of additional concreased K+ fluxes, stimulated by mitogen (13, 14). This ducting K+ channels. Voltage-gated K+ channels in squid giant axons have two-channel view of activation was supported by pharmacologic studies using Ca2+channel blockers to inhibit been directly shown to be permeable to Ca2+ in experimitogenesis ( 15 ) . and quinine, a blocker (albeit nonspe- ments using high Ca2+concentrations, passing about1 % cific) of calcium-activated K+ channels in other tissues, as much Ca++current as K+ current ( 18). This lack of to inhibit mitogen-induced membrane hyperpolarization perfect ion-selectivity is not unusual. Na+ channels also and K+ fluxes (13). However, Ca2+channels and Ca2+- exhibit a small degree of Ca2+permeability, and monoactivated K+ channels have not been observed in resting valent cations can pass through Ca2+channels ( 1 , 19). squid, or mitogen-activated voltage-clamped human T cells (3- Based upon the Ca2+permeability of K+ channels in 5 ) . Although it is difficult to rule out the possibility that we estimate that about 4000 Ca2+ ions per sec might Ca2+channels might be present under certain conditions,enter the T cell through each open K+ channel (5).Alvoltage-gated K+ channels are present in virtually all T though this level of permeability to Ca2+seems very low compared with that for K+, the calculated current carried cells. Consequently, our studies have led to a different hypothesis to account for the membrane hyperpolariza- by Ca2+through K+ channels is only about a n order of


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magnitude lower thanthecurrent carried by Ca2+ contrast, however, Birx et al. (22) reportthat verapamil through Ca2+channels atphysiologic levels of Ca2+(19), inhibits Con A-induced expression of the interleukin 2 partly because the conductance of K+ or Na+ channels is receptor on human lymphocytes. Allogeneic mixed lymmuch larger than that of Ca2+channels. If mitogen or phocyte stimulation is also inhibitedby K+ channel blockantigen opened one additional K+ channel, the intracel- ers (4AP, TEA, quinine) (ZO), as is the stimulation of the lular-free Caz+concentration in the absence of buffering D l 0 T cell line, either by Con A or by a clonospecific would double inabout 3 sec; due to cytoplasmic buffering antibody tothe T cell antigen receptor (inhibitedby 4AP. and diltiazem, unpublished the free Ca++levels would rise more slowly. According to TEA, quinine,verapamil this hypothesis, Ca” entry into the T cell through Kf studies in collaboration with Dr. C. A. Janeway, Yale channels would not depolarize the cell membrane be- University). Collectively, these data suggest that the K+ cause the netK+ channel currentwould be outward; this channel in T cells is required for early events leading up day is consistent with the observation that mitogens hyper- to protein synthesis which take place during the first during T cell activation. We are currently investigating polarize the membrane(13). are K+ channel-dependent. In collaboration with Dr. Roger Tsien (University of whether other early events A small fractionof human T cells express voltage-gated California, Berkeley)we used the Ca2+indicator dye, quin2, to test whether K+ channels in human T cells allow Na+ channels closely resembling sodium channels in Ca2+entry after mitogen stimulation. The intracellular- nerve and muscle. Sodium channels appear not to be free Ca2+concentration in resting Tcells was about 120 involved in T cell activation, because the specific Na+ nM and rose to about 230 nM after PHA addition. This channel blocker tetrodotoxin (TTX) had noeffect on mitrise in Ca2+was partially inhibited by pretreatment with ogenesis (5). In summary, K+ channels appearto play a vital role in concentrations of 4-aminopyridine (4AP) or verapamil sufficient to block most K+ channels. Addition of the T lymphocyte activation. K+ channels may function a s blockers after the PHA-induced rise in cytoplasmic-free signal transducers, linking receptors on the outside of T activation, Ca2+ reduced free Ca2+ partway toward resting levels. the cell with intracellular events during cell These results are consistent with the idea that at least or they may be required for other early stages of the part of the Ca2+entry into human T cells occurs through activation process. Cell killing. Voltage-gated K+ channels are present in open K+ channels. Electrically silent Ca2+entry pathways such as passive carriers or ion exchange mechanisms are clonal murine and in human cytotoxic T lymphocytes also a possibility. Thus, our studies on human T cells (24, unpublished data). Both Na+ and K+ channels are show that mitogens have amodulatory effect on K+ chan- present in two murine natural killer (NK) cell lines (unnel gating that can account for membrane hyperpolari- published studies incollaboration with Dr. Gunther Dennert, University of Southern California). Several lines of zation, K+ fluxes and at least part of the increase in evidence suggest that K+ channels arerequired for target cytosolic-free Ca2+during T cell mitogenesis. We have used a pharmacologic approach to determine cell lysis. Conjugation of cytotoxic T cells with target cells whether the K+ channel is necessary for mitogen-induced during the lethalhit phase is accompanied by increases in K+ conductance and 86Rbefflux (24).K+ channel blockproliferation. A number of chemically unrelatedsubstances, including “classical” blockersof K+ channels in ers reversibly inhibit targetcell lysis by human cytotoxic nerve and muscle (tetraethylammonium(TEA)and 4AP). T cells and by natural killer cells, in a dose-dependent Ca-activated K+ channel blockers (quinine and cetiedil). manner, at roughly the same concentrations that block a variety of calcium channel antagonists (verapamil, dil- the K+ channel in these cells: tetramethylammonium tiazem, nifedipine. nitrendipine, and several polyvalent (TMA),a n analog of TEA, neither blocks the K+ channel cations), calmodulin inhibitors (trifluoperazine and nor inhibits T cell killing (unpublished data). The exact chlorpromazine), and a @-receptorblocker with local an- role of the K+ channel in T cell and natural killer cellesthetic properties (propranolol), all blocked K+ channels mediated cytotoxicity remainsto be determined. Alin humanT cells, many at micromolar concentrations (3, though large sodium currents were present in clonal murine natural killer cells, the sodium channel blocker 20, 21, unpublisheddata). At similarconcentrations, those blockers that have been tested, namely,4AP, TEA. TTX had no effect on natural killer cell-mediated cytoquinine, verapamil, nifedipine,cetiedil and diltiazem, re- toxicity (unpublished data). versibly inhibited ”thymidine incorporation (3,20, 22, B lymphocytes and plasma cells. lnvolvement of ion unpublished data).In addition, propranolol and chlorpro- channels in B lymphocyte or plasma cell functions, inmazine inhibit PHA-induced 3H-thymidine incorporation cluding expression of activation antigens, capping, difin murine T cells (23). Inhibition of mitogenesis was not ferentiation, and antibody production and secretion, is due to nonspecific toxicity (3,20, 22, 23).Mitogen-stim- suggested by a large body of indirect evidence. A discusulated total protein synthesis was inhibited in a dose- sion of the correlations between ionic fluxes, membrane dependent mannerby the K+channel blockers 4AP, TEA, potential changes, increases in intracellular Caz+, and and quinine: interleukin 2production and secretion were varioustypes of activationparameters is beyond the also inhibited by 4AP. Block of mitogenesis by 4AP was scope of this review. Recent patch clamp studies reveal effective only if added during the first 20 to 30 hr of differences in the ion channel expression of B cells comstimulation with mitogen (20). Surprisingly, expression pared withT cells. Humanperipheral B lymphocytes of the interleukin 2 receptoris not inhibited by K+ chan- express K+ channels similar to those in human T cells, nel blockers (4AP. TEA, quinine) a t concentrations that but the numberof channels per cell seems tobe smaller inhibit protein synthesis (20). This result suggests that and more variable (unpublisheddata). the signals leading tothe expression of the interleukin2 A more dramatic difference between T and B derived receptor may not be mediated via the K+ channel. In cells is that voltage-gated Ca2+channels have been de-

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tected in the nonsecreting S I 9 4 myeloma cell line and in nel, sparingly permeable toCa2+(1 1).However, the great two immunoglobulin-secreting hybridoma B cell lines (8, concentration difference in Ca2+across the membrane 9). The density of Ca2+current in these cells increased may be sufficient to drive Ca2+ through the Fc-receptor after addition of fresh culture medium in parallel with channel, into the macrophage, providing a route for entry the secretion of immunoglobulin, but not withthe degree of extracellular Ca2+(11 , 27). In the absence of extracelof proliferation. The Ca2+channel antagonist D600 sup- lular Ca2+,macrophages are capable of phagocytosis of pressed both cell proliferation and immunoglobulin se- IgG-coated erythrocytes, at 40% of control levels (27). cretion, the latterto a greater extent. However, D600 was Buffering of intracellular-free Ca2+by quin-2/AM, supa rather weak Ca2+current blocker in these cells; thus, presses macrophagephagocytosis almost completely (27). the pharmacologic evidence does not strongly support a Thus, intracellular-free Caz+levels seem to be more imlink between Ca++channels and immunoglobulin secre- portant for phagocytosis than theextracellular Ca2+contion (9). Ca2' channelsin clonal hybridoma cells are centrations. Therole of Ca2+and theFc receptor channel insensitive tothe calcium channel antagonistsnifedipine ininterleukin1 synthesis and antigen processing reand nitrendipinea s well [Dr. S. Hagiwara, personal com- mains to be determined. munication). Surprisingly then, voltage-gated K+ chanImplications. In conclusion, the patchclamp technique nels in lymphocytes appear to be more sensitive toagents in combination with biochemical and immunologic methgenerally considered to be ea2+ channel antagonists thanods has provided a new approach to study cells of the are Ca2+channels in lymphocytes. immune system. The studies discussed in this article Macrophages. Recent studies with the gigaohm seal suggest a crucial role for ion channels in initiating, regtechnique have confirmed and extended earlier studies ulating, and supporting many cellular functions, includ(reviewed in Reference 25) suggesting that macrophages ing proliferation. Future studies using thisexciting new express ion channels. Human macrophages cultured for electroimmunologic approach may further clarify the link intracellularsignals several days display action potentials suggestingthe ex- between ion channelsandother istence of a n inwardcurrentchannel, presumably such as protein kinase-C, diacylglycerol and cyclic nupermeable eitherto Na+ or Ca2+ions (26).A voltage-gated cleotides, and therole played by ion channels in various K+ channel closely resembling the one in human T cells cellular functions and incell-cell communication. is present under some conditions in peritoneal mouse macrophages. No ion channels were detected 1 to 5 hr Acknowledgments. We gratefully acknowledge the after isolation,butnearlyall cells expressed delayed technical help of Ms. Mary Thornton andMr. Kevin Lee. rectifier K+ channels after 1 day in culture (6). A Ca2+REFERENCES activated K+ channel has been identified in human peripheral blood monocytes/macrophages cultured for 1 . Hille, B. 1984. Ionic Channels of Excitable Membranes. Sinauer Associates Inc.. Sunderland. MA. varying periods up to 3 wk (10).Different ion channels 2. Sakmann, B., and E. Neher. 1983.SingleChannel Recording. are expressed in the mouse macrophage cell line 5774.1 Plenum Press, New York. at various times after plating, during the process of ad3. Decoursey. T. E.,K. G. Chandy, S. Gupta, and M. D.Cahalan. 1984. Voltage-gated K+ channel in human T lymphocytes: a role in mitoherence (7).At first, no ion channels were detected. Outgenesis? Nature 307:465. ward K+ current through a delayed rectifier K+ channel 4. Matteson. D. R.. and C. Deutsch. 1984. K channels in T iymphoappeared2 to 6 hr after plating. Inward rectifier K+ cytes: a patch clamp study using monoclonal antibody adhesion. Nature 307:468. channels became prominent at later times, while the 5. Cahalan. M. D.,K. G. Chandy, T. E. DeCoursey. andS. Gupta. 1985. delayed rectifier K+ current diminished, reaching 0 at 24 A voltage-gated potassium channel in human T lymphocytes. J. Phystol. (Lond.)358:197. hr. Thereis some evidence that ion channels may play a 6. Ypey D. L., and D. E. Clapham. 1984. Development of a delayed role in phagocytosis and cytokine secretion by macrooutward-rectifying K+ conductance in cultured mouse peritoneal phages. Quinine, which blocks the delayed rectifier K+ macrophages. Proc. Natl. Acad. Scf. USA 81:3083. 7 . Sheehy, P. A.. and E. Gallin. 1984. Differential expression of two channel in T cells, has been reported to inhibit phagopotassium conductances in the macrophage-like cell line. 5774.1. cytosis by mouse peritoneal macrophages and the 5774 SOC. Neurosci. Abstr. 10:870. 8. Fukushima. Y.. and S. Hagiwara. 1983. Voltage-gated Ca2+channel macrophage cell line (27). This inhibitory effectmay be in mouse myeloma cells. Proc. Natl. Acad. Sci. USA 80:2240. mediated by blockade of macrophage-delayed rectifier K+ 9. Fukushima. Y., S. Hagiwara, andR. E. Saxton. 1984. Variations of channels, or of calcium-activated K+ channels. Prelimicalcium current during the cell growth cycle in mouse hybridoma lines secreting immunoglobulins. J. Physiol. (Lond.).355:313. nary studies in our laboratory indicate that interleukin 1 10. Gallin, E. K. 1984. Calcium- and voltage-activated potassium chansynthesis by human peripheral blood monocyte/macronels in human macrophages. Bfophys. J.46:821. phages is inhibited by the K+ channel blocker 4AP, in a 1 1 . Young, J. D. E., J. C. Unkeless, H. R. Kaback. and 2. A. Cohn. 1983. Role for mouse macrophage 1gG Fc receptor as ligand-dependdose-dependent manner. ent ion channel. Nature 306:186. Interaction of ligands (e.g., IgG-coated erythrocytes) 12. Mastro, A. M., and M. C. Smith. 1983. Calcium-dependentactivation of lymphocytes by ionophore, A23187. and a phorbol ester tumor with the mouse macrophage Fc receptor transiently inpromoter. J. Cell. Physiol. 1 16:5 1 . creases cytosolic-free Ca2+,triggers phagocytosis and the 13. Tsien, R. Y., T. Pozzan. and T. Rink. 1982. T-cell mitogens cause secretion of various mediators of inflammation, and inearly changes in cytoplasmic free Ca2+and membrane potential in lymphocytes. Nature 295938. creases Na+/K+ fluxes across the membrane [see Refer14. Metcalfe, J. C., T. Pozzan, G. A. Smith, andT. R. Hesketh. 1980. A ences 11 and 27). The elevation in intracellular freeCa2+ calcium hypothesis for the control cell growth. Biochem. Soc. Symp. 45: 1. is only partially (70%)inhibited by the absenceof extraE., and T. Diamantstein. 1978. Inhibition by cellular Ca2+,suggesting releaseof Ca2+from intracellular 15. Blitstein-Willinger, lsoptin (a calcium antagonist) of the mitogenic stimulation of lymstores, in addition to a n influx from the outside (27). phocytes prior to the S phase. Immunology 34:303. Purified mouse macrophage Fc receptor has been incor- 16. DeCoursey, T. E., K. G. Chandy. M. Fischbach. N. Talal, S. Gupta. and M.D. Cahalan. 1985. Two types of K channels in T lymphocytes porated into planar lipid bilayers and shown to form a from MRL mice. Biophys. J. 47:387a. ligand-dependent monovalent cation-selective ion chan- 17. Owens, T., and J. G. Kaplan. 1980. Increased cationic ion fluxes in


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stimulated lymphocytes of the mouse: response of enriched B- and T-cell subpopulations to 8-and T-cell mitogens. Can. J. Blochern. Cell Bid. 58~831. Inoue, 1. 1981. Activation-inactivation of potassium channels and development of the potassium-channel spike ininternally perfused squid giant axons. J. Gen. Physlol. 78r43. H e s s . P.. and R. W. Tsien. 1984. Mechanism of ion permeation through calcium channels. Nature309:453. Chandy, K. G.. T. E. Decoursey, M. D. Cahalan. C. McLaughlin. and S. Gupta. 1984. Voltage-gated K channels are required for T lymphocyte activation. J. Exp. Med. 160:369. Decoursey, T. E.,K. G. Chandy, S.Gupta, and M. D.Cahalan. 1984. Pharmacology of human T lymphocyte K channels. Blophys. J . 45: 144Q. Birx. D. L., M. Berger, and T. A. Fleisher. 1984. Theinterference of T cell activation by calcium channel blocking agents. J . Irnrnunol.

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133:2904. 23. Anderton, B. H., J. S. Axford, P. Cohn. N. J. Marshall, L. Shen. and S.Sprake. 198 1. Inhibition of lymphocyte capping and transformation by propranolol and related compounds.Br. J. Pharrnacol. 72:69. 24. Fukushima, Y.. S. Hagiwara.and M. Henkart. 1984. Potassium current in clonal cytotoxic T lymphocytes from the mouse. J. Physiol. (Lond)351:645. 25. Gallin, E. K. 1984. Electrophysiological properties of macrophages. Fed. Proc. 43:2385. 26. McCann, F. V., J. J. Cole, P. M. Guyre, and J. A. G. Russell. 1983. Action potentials in macrophages derived from human monocytes. Science. 219:191. 27. Young, J. D. E., S.S. KO,2. A. Cohn. 1984. The increase in intracellular free calcium associated with IgG 2b/l Fc receptor-ligand interactions: role in phagocytosis. Proc. Natl. Acad. Scl. LISA 81:5430.