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Calcium-activated potassium channels in lymphocytes
Cell Calcium 4:
463-473, 1983
CALCIUM-ACTIVATEDPOTASSIUM CHANNELSIN LYMPHOCYTES Timothy J. Rink and Carol Deutsch* Physiological Laboratory, Downing Street, Cambridge University CB2 3EG, U.K., and *Department of Physiology, of Pennsylvania, Philadelphia 19104, U.S.A. (Reprint requests to TJR.) INTRODUCTION This review summarizes the available evidence that some types of lymphocytes, have calcium-activated So far the main indication for the potassium channels. presence of these channels has come from: (1) indirect measurements of membrane potential using various chemical of 86Rb fluxes. The evidence probes , and (2) measurements may seem sparse and incomplete but we should point out that it has mostly come as a by-product of experiments concerned with other matters rather than those aimed primarily at demonstrating the existence and properties of calcium-activated potassium channels. It is also important to emphasise that there are many sub-populations which may have different properties and of lymphocytes! The major there can be significant species variations. sub-divisions are B and T-cells. B-cells are mainly concerned with production and secretion of antibodies. T-cells are functionally heterogeneous playing a variety induction of activity for effector cytotoxicity, of roles: functions (i.e. helper cells) and suppression of these Many studies have used crude preparations of functions. lymphocytes such as those isolated from peripheral blood, or pig mesenteric lymph nodes. and rodent thymus, spleen, All these contain many sub-populations of lymphocytes as well as a proportion of other cells such as macrophages. Clearly the interpretation of measurements on such preparations may be complicated by functional heterogeneity. Many technical problems may be by-passed by the use of the patch-clamp technique on single cells. Very recently Matteson and Deutsch [l] have made patch-clamp recordings of currents from antigenically identified lymphocytes. A voltage-dependent potassium channel was found but, as yet, there is no evidence 463
concerning activation by calcium ions. This powerful electrophysiological tool will no doubt soon provide much data on the membrane properties of different types of lymphocytes at different sta es of maturity. In the meantime we offer a review 0+ the presently available more indirect data from which inferences about calciumdependent potassium channels can be drawn. ION DISTRIBUTION In order to interpret measurements of membrane potential or ion flux one needs values for concentrations of the major ions. Fortunately, cytoplasmic levels of the ions are fairly well documented for the major inorganic resting lymphocytes and the values are typical of those found in most cells. The potassium and sodium contents are around 140 and 20 mmol/l cell water respectively [e.g. This distribution of potassium and sodium is 2-81. maintained by the usual sodium pump and the gradients of these ions dissipate over a matter of hours if the. sodium pump is inhibited by ouabain. The intracellular chloride content is about 35 mmol/l cell water [9,10]. Cytoplasmic pH is normally well stabilized near about 7.1 [112:2]. Free Mg2+ is around 0.9 mM [12] and basal free Ca is near 0.1 uM [13-151. MEMBRANEPOTENTIAL Direct electrophysiological measurements of lymphocyte membrane potential have been reported [16,17]. due to impalement The low values c. -10 mV were probably 7-10 urn, high impedance cells. These damage of the small, which were made many years ago, used relatively studies, coarse microelectrodes. To our knowledge there are no studies in which up-to-date ultrafine electrodes, or methods which allow recording of the potential immediately Indirect measurements after impalement have been used. using various chemical probes give values for resting in lymphocytes between -50 and -7UmV [6,7,13,18-201, reasonable agreement considering the possible artefacts of these indirect techniques [23] and the different cell The probes that have been used preparations employed. include two fluorescent dyes, diS-C3-(5) and bis-(1,3_diethylthiobarbiturate) trlmethine-oxonol, and the radio labelled lipophylic cations tetraphenylIn ion phosphonium and triphenylmethylphosphonium. substitution experiments the measured potential is found to be most sensitive to increases in potassium and less sensitive to substitution of sodium or chloride by larger, Most workers have therefore supposedly impermeant ions. concluded that the membrane is relatively selective to Nonetheless the resting potential is potassium. 464
apparently indicating
some 20-30 mV depolarized a significant permeability
from E presumably to ot k’er ions.
In many preparations, the first hint of the presence of calcium-activated potassium channels has come from observing a hyperpolarization (and perhaps a decreased input impedance) under conditions where [Ca2+]i is In lymphocytes, the expected or known to be increased. first clue came from finding a depolarization under conditions in which it was guessed that calcium-activated It was observed that potassium channels would be blocked. the membrane potential of mouse spleen lymphocytes became less negative with indicated by diS-C3-(51, increasing dye concentration especially in the presence of low external potassium [6]. This effect was reminiscent of the way that this and the related dye blocked calcium particularly at low external activated potassium channels, potassium, in red cell ghosts [24]. Furthermore, quinine which was also known to block calcium-activated potassium channel in red blood cells [ 25 I, depolarized lymphocytes in a manner consistent with blockade of a potassium channel [ 61. It has also been reported that quinine depolarizes rodent thymocytes and pig mesenteric lymph nodes [7]. One explanation for the depolarizing effect of quinine could be that part of the basal potassium conductance is due to calcium-activated channels and is reduced by the drug. This would be unusual as such channels are thought normal1 to be activated by However, there are stimulated elevations of [Ca ?z+]i* other possible explanations. Quinine cannot be regarded as specific for calcium-activated channels and it may block other forms of potassium conductance. For 100 uM quinine has direct effects on the instance, voltage-dependent potassium channel observed in Another possibility is that in the lymphocytes [ 11. conditions of these experiments [Ca2+]. was somewhat elevated and the membrane was therefor; more than normally permeable to potassium. The measurements on mouse spleen cells [6] were done with DiS-C3-(5) and those on rodent thymocytes and pig mesenteric node cells were mostly done with TPMP in the presence of FCCP [22]. DiS-C3-(5) [23] and FCCP are potential mitochondrial poisons and can [13,14]. Continuing this elevate [ Ca2+] i in lymphocytes approach, Felber and Brand have used the ability of quinine to depolarize lymphocytes, assessed with TPMP or the oxonol dye, as an indication of the presence of calcium-activated potassium channels in lymphocytes from pig mesenteric nodes and rodent thymus. Because lymphocyte membrane potential is somewhat depolarized from Ek one might expect 3 hyper olarization following deliberate elevation of [Ca +]i, w g ich should increase the potassium permeability. In the first reports of calcium ionophores actually [e.g. 61, application 465 (‘C
G
appear to depolarize lymphocytes. It seem likely that this was due to excessively high levels, 2-4 uM A23187, that apparently discharged the sodium_ and potassium gradients either by raising [Ca2+]* to toxic levels or some other ill effect. More modest elevations of [Ca2+]. from the normal basal level of near 120 nm to about 1 uM1 did hyperpolarize mouse thymocytes assessed by oxonol fluorescence and this hyperpolarization is blocked by 200 uM quinine [13]. In pig mesenteric node lymphocytes, calcium ionophore appears not to hyperpolarize the membrane potential [22] even at concentrations chosen to give the same increase in [Ca2+]i that does depolarize thymocytes [Rink, T.J. and Tsien, R.Y., unpublished observations]. One could interpret this either as being due to the absence of calcium activated potassium channels or to the fact that they are fully open in the basal condition. Felber and Brand [22] support the latter possibility on the basis of the depolarization caused by 0.5 mM quinine. 86Rb FLUXES The evidence just discussed in favour of the existence of calcium activated potassium channels is very indirect, being based on membrane potential measurements using chemical probes. In the absence of more direct data it is difficult to exclude the possibility that at least some of the changes in membrane potential described above could have affected changes in other types of potassium channels or even changes in the permeability to sodium or other ions. Somewhat more direct evidence for calcium activated potassium channels in human peripheral blood lymphocytes and rabbit thymocytes has been obtained by for potassium measurements of 86Rb flux as a tracer In the presence of external calcium, A23187 movements. caused a several-fold increase in 86Rb efflux and uptake In the absence of external calcium the [ 10, 26-281. fluxes were little altered. The ionophore-induced 86Rb flux was greatly reduced by 15 uM quinine, which had little effect on the control fluxes. These data support the existence of a Ca-activated potassium permeability in these cells, and one that is far from fully activated in resting conditions. The ionophore activated 86Rb fluxes were also inhibited by 10 uM trifluoperazine which might indicate a role for calmodulin, although as the authors pointed out trifluoperazine is not a very selective In pig lymphocytes A23187 did not inhibitor 261. Q6Rb flux, but quinine did decrease it 1221. stimulate
466
PROPERTIES
OF CALCIUM ACTIVATED POTASSIUM CHANNELS
Since the evidence for even the existence of these channels is so sketchy it is not surprising that there is little information on their mechanism or activation. In only one study have both [Ca”]i and membrane potential From those data it appears that been measured [13]. potassium channels in mouse thymocytes are activated by [Ca2+]i in the region of 1 uM. The Gardos channel of human red cells has a requirement for external potassium [25] and the same may be true for lymphocytes. The hyperpolarization of mouse thymocytes normally activated by modest elevation of a2+]* appeared to be abrogated in potassium-free LZlutihns although the resting potential was not much altered [Rink, T.J. unpublished observations]. This probably did not reflect an effect of inhibition of the sodium pump as a dose of ouabain sufficent to block the pump even in rodent cells did not have the same effect. A recent attempt to investigate calcium-activated potassium channels in inside-out vesicles of the plasma membrane of the rabbit thymocytes was unfortunately unsuccessful in that no calcium or calmodulin dependence of 86Rb fluxes could be found [28]. The authors suggested that the lack of responsiveness might reflect the absence of essential phosphorylation of a component of the isolated membranes rather than the absence in the original membrane of calcium activated channels. VOLUMEREGULATIONAND POTASSIUMPERMEABILITY Human peripheral lymphocytes, and rabbit thymocytes, respond to hypotonic swelling with rapid volume restoration achieved by increased permeability to potassium, and to chloride, with consequent loss of cellular KC1 [10,14,16-321. The enhanced potassium flux shares some properties with that induced by A23187 including blockade by quinine. However, other properties of the two potassium permeabilities are rather different including the sensitivity to trifluoperazine and the 86Rb fluxes requirement for external calcium [26,31]. stimulated by A23187 are largely dependent on external calcium. The volume regulation does not require the presence of external calcium, although a prolonged incubation in calcium free medium inhibits the volume restoration. Recent studies with the calcium indicator quin2 show that hypotonic swelling did not alter [Ca2+]i and that volume restoration occurred while calcium remained unchanged at basal levels [15]. It seems therefore that the swellin increase in potassium -induced permeability is activated t y some means other than elevated [Ca2+]i. Further study will be needed to see if 467
the two conditions same channel or to potassium channel.
lead to a different activation activation of different types
of of
the
MITOGEN-INDUCEDALTERATION IN POTASSIUM PERMEABILITY There are numerous reports of increased cation fluxes following mitogenic stimulation of lymphocytes [2,9,13,33-431. The findings have varied according to the preparation and methods used, the amount and type of mitogen and the period of observation. In many cases an increased potassium flux has been observed. In the recent patch-clamp study there was a c. 2-fold enhancement of potassium currents in mitogen-stimulated human lymphocytes In some instances there is evidence that mitogens Ill. may stimulate a calcium-activated potassium conductance. The mitogenic lectin, concanavalin A, was found to cause an early elevation of [Ca2+1- and a concurrent hyperpolarization in mouse tAymocytes [13]. This response was dependent on external calcium and blocked by quinine but not by ouabain. Therefore the concanavalin A-induced response was consistent with activation of potassium channels by the elevated [Ca2+]i. A similar hyperpolarization of mouse thymocytes was observed by Felber and Brand [22], who also reported that concanavalin A increased 86Rb influx. In pig lymphocytes, however, concanavalin A raises [Ca 2+]i [13] but apparently dggibnot cause a measurable hyperpolarization, or increased As discussed in connection with the failure of flux [22]. calcium ionophore to hyperpolarize pig cells, this could be due to the presence of a fully activated calcium-activated potassium conductance, or to the absence Studies of mitogenic of such a conductance. transformation in human peripheral lymphocytes have given conflicting estimates of membrane potential changes; some authors reporting a depolarization [e.g. 211, others no In preliminary measurements significa;t change [e.g. 191. A produced no with quin , it was found that concanavalin measurable change in [Ca2+]i in human peripheral blood observation] in lymphocytes [Rink, T. J., unpublished contrast to the results with mouse thymocytes and pig mesenteric node lymphocytes. The significance of the hyperpolarization induced by One concanavalin A in thymocytes remains obscure. possible role for a hyperpolarization of the membrane is that this could help drive an increased sodium-linked uptake of organic substance such as glucose and amino have suggested that acids. However, two groups [19,221 membrane potential plays no critical role in the response to lectins at least in the first two to three hours of stimulation.
468
CONCLUSION The finding
calcium ionophore can produce a 86Rb flux in several lymphocyte preparations presently provides the strongest evidence for the existence of calcium-activated potassium channels. The presence of such channels is also consistent with the observations that (i) elevations of [Ca*+Ii by ionophore or mitogenic lectins can hyperpolarize some types of and (ii) that substances known to block these lymphocytes, channels in other cells can prevent this hyperpolarizaand can depolarize the resting membranes of some tion, It seems likely that there are several kinds lymphocytes. of potassium channel in lymphocyte membranes and that not all lymphocytes have the same membrane properties. It is expected that application of modern electrophysiological techniques particularly the patch-clamp in its various forms will soon allow a detailed analysis of the electrical properties of defined sub-populations of lymphocytes. This should resolve many questions about the existence, nature and functions of these channels. specific
increase
that in
ACKNOWLEDGEMENTS The authors ’ work has been supported by grants from the SERC, UK, and the NIH, U.S.A. C.D. holds a Research Career Development Award. We thank Dr. M. Brand and MS S. Felber for helpful discussion. REFERENCES 1.
Matteson, D.R. and Deutsh, C. K channels T-lymphocytes. A patch clamp study using antibody adhesion. MS submitted
2.
Segel, G.B., Simon, W. and Lichtman, M.A. (1979). Regulation of sodium and potassium transport in phytohemagglutinin-stimulated human blood lymphocytes. Journal of Clinical Investigation, 834-841.
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Lichtman, M.A., Jackson, A.H. and Peck, W.A. (1972). Lymphocyte monovalent cation metabolism: cell volume, cation content and cation transport. Journal of Cellular Physiology 80,383-396
4.
Negendank, W.G. and Collier, C.R. (1976) Ion contents of human lymphocytes. The effects of concanavalin A and ouabain. Experimental Cell Research 101, 31-40
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Negendank, W. and Shaller, C. (1979). Fast and slow fractions of K+ flux in human lym hocytes. Journal of Cellular Physiology 98, 539-555;.
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Rink, T.J., Montecucco, C., Hesketh, T.R. and Tsien, R.Y. (1980) Lymphocyte membrane potential assessed with fluorescent probes. Biochimica et Biophysics Acta 595, 15-30
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Felber, S.M. and Brand, M.D. (1982) Factors determining the plasma-membrane potential of lymphocytes. Biochemical Journal 204, 577-585
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Felber, S.M. and Brand, M.D. (1983) Concanavalin causes an increase in sodium permeability and intracellular sodium content of pig lymphocytes. Biochemical Journal 210, 893-897
9.
Iversen, J.G. (1976) Unidirectional K+ fluxes in rat thymocytes stimulated by concanavalin A. Journal of Cell Physiology 89, 262-276
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Grinstein, S., Clarke, C.A. and Rothstein, N. (1982) Increased anion permeability during volume regulation Philosophical Transactions of in human lymphocytes. the Royal Society B 299, 509-518.
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Deutsch, C., Taylor, J.S. and Wilson, D.F. (1982) Regulation of intracellular pH by human peripheral blood lymphocytes as measured by lgF NMR. Proceedings of the National Academy of Sciences, U.S.A. 79, 7944-7948
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Rink, T.J., Tsien, R.Y. and Pozzan, T. (1982) Journal Cytoplasmic pH and free M 2+ in lymphocytes. of Cell Biology 95, la9-186
13.
Tsien, R.Y., Pozzan, T. and Rink, T.J. (1982) T-cell mitogens cause early changes in cytoplasmic free Ca2+ Nature 295, and membrane potential in lymphocytes. 68-71
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Tsien, R.Y., Pozzan, T. and Rink, T.J. (1982) Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new intracellularly trapped Journal of Cell Biology 94, fluorescent indicator. 325-334
15.
Rink, T.J., Sanchez, A., Grinstein, S. and Rothstein, A. (1983) Volume restoration in osmotically swollen lymphocytes does not involve changes in free Ca2+ Biochimica et Biophysics Acta 762, concentration. 593-596
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Taki, M. (1970) Studies on blastogenesis of human lymphocytes by phytohaemaglutinin, with special reference to changes of membrane potential during blastoid transformation. Mie Medical Journal 19, 245-262
17.
Malofiejew, M., Kostrzewska, A. and Kowal, E. (1975) Messungen elektrisher membranpolentrale von lymphozyten. Acta Biologica et Medica Germanica 34, 1007-1011.
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Deutsch, C.J., Holian, S.K., Holian, P., Daniele, P. and Wilson, D.F. (1979) Transmembrane electrical and pH gradient, across human erythrocytes and human peripheral lymphocytes. Journal of Cellular Physiology 99, 79-94
19.
Deutsch, C. and Price, M. (1982) Role of extracellular Na and K in lymphocyte activation. Journal of Cellular Physiology 111, 73-79.
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Kiefer, H. Blume, A. and Kaback, R. (1980). Membrane potential changes during mitogenic stimulation of mouse spleen lymphocytes. Proceedings of the National Academy of Sciences, U.S.A. 77, 2200-2204.
21.
Shapiro, H.M., Natale, P. and Kamentsky, L.A. (1979). Estimation of membrane potentials of individual lymphocytes by flow cytometry. Proceedings of the National Academy of Sciences, U.S.A. 76, 5728-2730.
22.
Felber, S.M. and Brand, M.D. (1983) Early plasmamembrane-potential changes during stimulation of lymphocytes with concanavalin A. Biochemical Journal 210, 885-891
23.
Rink, T.J. (1982). Measurement of membrane potential with chemical probes. In Techniques in the Life Sciences, B4/II, Lipid and Membrane Biochemistry B423, pp. l-29. Elsevier Scientific Publishers, Ireland.
24.
Simons, T.J.B. (1976) Carbocyanine dyes inhibit Cadependent K+ efflux from human red cell ghosts. Nature 264, 467-469
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Lew, V.L. transport potassium Topics in
26.
Grinstein, S., Dupre, A. and Rothstein, Volume regulation in human lymphocytes: calcium. Journal of General Physiology
and Ferreira, M.G. (1978) Calcium and the properties of a calcium-activated channel in red cell membranes. Current Membrane Transport 10, 217-277
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Cheung, R.K., Grinstein, S., Bosch, H.M. and Gelfand, E.W. (1982) Volume regulation by human lymphocytes. Characterisation of the ionic basis for regulatory volume decrease. Journal of Cellular Physiology 112, 189-196.
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Grinstein, S., Cohen, S. Sarkadi, B. and Rothstein, A. (1983) Induction of g6Rb fluxes by Ca2+ and volume changes in thymocytes and their isolated membranes. Journal of Cellular Physiology 46. In press
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Doljanski, F., Ben-Sasson, S., Reich, M. and Grover, N.B. (1974). Dynamic osmotic behavior of chick blood lymphocytes. Journal of Cellular Physiology 84, 215-224.
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Ben-Sasson, S., Shaviv, R., Bentwich, Z., Slavin, S. and Doljanski, F. (1975). Osmotic behavior of normal and leukemic lymphocytes. Blood 46, 891-899.
31.
Bui, A.H. and Wiley, J.B. (1981). Calcium fluxes and volume regulation in human lymphocytes. Journal of Cellular Physiology 108, 47-54.
32. Deutsch, C., Slater, L. and Goldstein, P. (1982) Volume regulation of human peripheral blood lymphocytes and stimulated proliferation of volume adanted cells. Biochimica et Bionhvsica Acta 721, L r 262-267 33.
Quastel, M.R. and Kaplan, J.G. (1970). Early stimulation of potassium uptake in lymphocytes treated with PHA. Experimental Cell Research 63 230-233.
34.
Averdunk, R. and Lauf, P.K. (1975). Effzcts of mitogens on sodium-potassium transport, jH:ouabain binding and adenosine triphosphatase activity In lymphocytes. Experimental Cell Research 93, 331-342.
35.
Segel, G.B., Gordon, B.R., Lichtman, M.A., Hollander, M M. and Klemperer, M.R. (1976). Exodus of 42K+ and g&Rb from rat th mic and human blood 1 mphocytes exposed to phytoz emagglutinin. JournaP of Cellular Physiology 87, 337-344.
36.
G.B. and Lichtman, M.A. (1976). Potassium Segel, transport in human blood lymphocytes treated with phytohemagglutinin. Journal of Clinical Investigation 58, 1358-1369.
37.
Hamilton, L.J. and Kaplan, J.G. (1977). Flux of g6Rb in activated human lymphocytes. Canadian Journal of Biochemistry 55, 774-778. 472
38.
Averdunk, R. and Gunther, T. (1980). Effect of concanavalin A on intracellular K+ and Na+ concentration and K+ transport of human lymphocytes. Immunobiology 157, 132-144.
39. Deutsch, C. and Price, M. (1982). Cell calcium in human peripheral blood lymphocytes and the effect of mitogen. Biochimica et Biophysics Acta 687, 211-218. 40.
Whitney, R.B. and Sutherland, R.M. (1972). Characteristics of calcium accumulation by lymphocytes and alterations in the process induced by phytohemagglutinin. Journal of Cellular Physiology 82, 9-20.
41.
Allwood, G., Asherson, G.L., Davey, M.J. and Goodferd, P.J. (1971). The early uptake of radioactive calcium by human lymphocytes treated with phytohemagglutinin. Immunology, 21, 509-516.
42.
Freedman, M.H., Raff, M.C. and Gomperts, B. (1975). Induction of increased calcium uptake in mouse T lymphocytes by concanavalin A and its modulation by cyclic nucleotides. Nature 255, 378-382.
43.
Parker, C.W. (1974). Correlation between mitogenicity and stimulation of calcium uptake in human lymphocytes. Biochemical and Biophysical Research Communications 61, 1180-1186.
Received Accepted
3.10.83 2.11.83
473
463-473, 1983
CALCIUM-ACTIVATEDPOTASSIUM CHANNELSIN LYMPHOCYTES Timothy J. Rink and Carol Deutsch* Physiological Laboratory, Downing Street, Cambridge University CB2 3EG, U.K., and *Department of Physiology, of Pennsylvania, Philadelphia 19104, U.S.A. (Reprint requests to TJR.) INTRODUCTION This review summarizes the available evidence that some types of lymphocytes, have calcium-activated So far the main indication for the potassium channels. presence of these channels has come from: (1) indirect measurements of membrane potential using various chemical of 86Rb fluxes. The evidence probes , and (2) measurements may seem sparse and incomplete but we should point out that it has mostly come as a by-product of experiments concerned with other matters rather than those aimed primarily at demonstrating the existence and properties of calcium-activated potassium channels. It is also important to emphasise that there are many sub-populations which may have different properties and of lymphocytes! The major there can be significant species variations. sub-divisions are B and T-cells. B-cells are mainly concerned with production and secretion of antibodies. T-cells are functionally heterogeneous playing a variety induction of activity for effector cytotoxicity, of roles: functions (i.e. helper cells) and suppression of these Many studies have used crude preparations of functions. lymphocytes such as those isolated from peripheral blood, or pig mesenteric lymph nodes. and rodent thymus, spleen, All these contain many sub-populations of lymphocytes as well as a proportion of other cells such as macrophages. Clearly the interpretation of measurements on such preparations may be complicated by functional heterogeneity. Many technical problems may be by-passed by the use of the patch-clamp technique on single cells. Very recently Matteson and Deutsch [l] have made patch-clamp recordings of currents from antigenically identified lymphocytes. A voltage-dependent potassium channel was found but, as yet, there is no evidence 463
concerning activation by calcium ions. This powerful electrophysiological tool will no doubt soon provide much data on the membrane properties of different types of lymphocytes at different sta es of maturity. In the meantime we offer a review 0+ the presently available more indirect data from which inferences about calciumdependent potassium channels can be drawn. ION DISTRIBUTION In order to interpret measurements of membrane potential or ion flux one needs values for concentrations of the major ions. Fortunately, cytoplasmic levels of the ions are fairly well documented for the major inorganic resting lymphocytes and the values are typical of those found in most cells. The potassium and sodium contents are around 140 and 20 mmol/l cell water respectively [e.g. This distribution of potassium and sodium is 2-81. maintained by the usual sodium pump and the gradients of these ions dissipate over a matter of hours if the. sodium pump is inhibited by ouabain. The intracellular chloride content is about 35 mmol/l cell water [9,10]. Cytoplasmic pH is normally well stabilized near about 7.1 [112:2]. Free Mg2+ is around 0.9 mM [12] and basal free Ca is near 0.1 uM [13-151. MEMBRANEPOTENTIAL Direct electrophysiological measurements of lymphocyte membrane potential have been reported [16,17]. due to impalement The low values c. -10 mV were probably 7-10 urn, high impedance cells. These damage of the small, which were made many years ago, used relatively studies, coarse microelectrodes. To our knowledge there are no studies in which up-to-date ultrafine electrodes, or methods which allow recording of the potential immediately Indirect measurements after impalement have been used. using various chemical probes give values for resting in lymphocytes between -50 and -7UmV [6,7,13,18-201, reasonable agreement considering the possible artefacts of these indirect techniques [23] and the different cell The probes that have been used preparations employed. include two fluorescent dyes, diS-C3-(5) and bis-(1,3_diethylthiobarbiturate) trlmethine-oxonol, and the radio labelled lipophylic cations tetraphenylIn ion phosphonium and triphenylmethylphosphonium. substitution experiments the measured potential is found to be most sensitive to increases in potassium and less sensitive to substitution of sodium or chloride by larger, Most workers have therefore supposedly impermeant ions. concluded that the membrane is relatively selective to Nonetheless the resting potential is potassium. 464
apparently indicating
some 20-30 mV depolarized a significant permeability
from E presumably to ot k’er ions.
In many preparations, the first hint of the presence of calcium-activated potassium channels has come from observing a hyperpolarization (and perhaps a decreased input impedance) under conditions where [Ca2+]i is In lymphocytes, the expected or known to be increased. first clue came from finding a depolarization under conditions in which it was guessed that calcium-activated It was observed that potassium channels would be blocked. the membrane potential of mouse spleen lymphocytes became less negative with indicated by diS-C3-(51, increasing dye concentration especially in the presence of low external potassium [6]. This effect was reminiscent of the way that this and the related dye blocked calcium particularly at low external activated potassium channels, potassium, in red cell ghosts [24]. Furthermore, quinine which was also known to block calcium-activated potassium channel in red blood cells [ 25 I, depolarized lymphocytes in a manner consistent with blockade of a potassium channel [ 61. It has also been reported that quinine depolarizes rodent thymocytes and pig mesenteric lymph nodes [7]. One explanation for the depolarizing effect of quinine could be that part of the basal potassium conductance is due to calcium-activated channels and is reduced by the drug. This would be unusual as such channels are thought normal1 to be activated by However, there are stimulated elevations of [Ca ?z+]i* other possible explanations. Quinine cannot be regarded as specific for calcium-activated channels and it may block other forms of potassium conductance. For 100 uM quinine has direct effects on the instance, voltage-dependent potassium channel observed in Another possibility is that in the lymphocytes [ 11. conditions of these experiments [Ca2+]. was somewhat elevated and the membrane was therefor; more than normally permeable to potassium. The measurements on mouse spleen cells [6] were done with DiS-C3-(5) and those on rodent thymocytes and pig mesenteric node cells were mostly done with TPMP in the presence of FCCP [22]. DiS-C3-(5) [23] and FCCP are potential mitochondrial poisons and can [13,14]. Continuing this elevate [ Ca2+] i in lymphocytes approach, Felber and Brand have used the ability of quinine to depolarize lymphocytes, assessed with TPMP or the oxonol dye, as an indication of the presence of calcium-activated potassium channels in lymphocytes from pig mesenteric nodes and rodent thymus. Because lymphocyte membrane potential is somewhat depolarized from Ek one might expect 3 hyper olarization following deliberate elevation of [Ca +]i, w g ich should increase the potassium permeability. In the first reports of calcium ionophores actually [e.g. 61, application 465 (‘C
G
appear to depolarize lymphocytes. It seem likely that this was due to excessively high levels, 2-4 uM A23187, that apparently discharged the sodium_ and potassium gradients either by raising [Ca2+]* to toxic levels or some other ill effect. More modest elevations of [Ca2+]. from the normal basal level of near 120 nm to about 1 uM1 did hyperpolarize mouse thymocytes assessed by oxonol fluorescence and this hyperpolarization is blocked by 200 uM quinine [13]. In pig mesenteric node lymphocytes, calcium ionophore appears not to hyperpolarize the membrane potential [22] even at concentrations chosen to give the same increase in [Ca2+]i that does depolarize thymocytes [Rink, T.J. and Tsien, R.Y., unpublished observations]. One could interpret this either as being due to the absence of calcium activated potassium channels or to the fact that they are fully open in the basal condition. Felber and Brand [22] support the latter possibility on the basis of the depolarization caused by 0.5 mM quinine. 86Rb FLUXES The evidence just discussed in favour of the existence of calcium activated potassium channels is very indirect, being based on membrane potential measurements using chemical probes. In the absence of more direct data it is difficult to exclude the possibility that at least some of the changes in membrane potential described above could have affected changes in other types of potassium channels or even changes in the permeability to sodium or other ions. Somewhat more direct evidence for calcium activated potassium channels in human peripheral blood lymphocytes and rabbit thymocytes has been obtained by for potassium measurements of 86Rb flux as a tracer In the presence of external calcium, A23187 movements. caused a several-fold increase in 86Rb efflux and uptake In the absence of external calcium the [ 10, 26-281. fluxes were little altered. The ionophore-induced 86Rb flux was greatly reduced by 15 uM quinine, which had little effect on the control fluxes. These data support the existence of a Ca-activated potassium permeability in these cells, and one that is far from fully activated in resting conditions. The ionophore activated 86Rb fluxes were also inhibited by 10 uM trifluoperazine which might indicate a role for calmodulin, although as the authors pointed out trifluoperazine is not a very selective In pig lymphocytes A23187 did not inhibitor 261. Q6Rb flux, but quinine did decrease it 1221. stimulate
466
PROPERTIES
OF CALCIUM ACTIVATED POTASSIUM CHANNELS
Since the evidence for even the existence of these channels is so sketchy it is not surprising that there is little information on their mechanism or activation. In only one study have both [Ca”]i and membrane potential From those data it appears that been measured [13]. potassium channels in mouse thymocytes are activated by [Ca2+]i in the region of 1 uM. The Gardos channel of human red cells has a requirement for external potassium [25] and the same may be true for lymphocytes. The hyperpolarization of mouse thymocytes normally activated by modest elevation of a2+]* appeared to be abrogated in potassium-free LZlutihns although the resting potential was not much altered [Rink, T.J. unpublished observations]. This probably did not reflect an effect of inhibition of the sodium pump as a dose of ouabain sufficent to block the pump even in rodent cells did not have the same effect. A recent attempt to investigate calcium-activated potassium channels in inside-out vesicles of the plasma membrane of the rabbit thymocytes was unfortunately unsuccessful in that no calcium or calmodulin dependence of 86Rb fluxes could be found [28]. The authors suggested that the lack of responsiveness might reflect the absence of essential phosphorylation of a component of the isolated membranes rather than the absence in the original membrane of calcium activated channels. VOLUMEREGULATIONAND POTASSIUMPERMEABILITY Human peripheral lymphocytes, and rabbit thymocytes, respond to hypotonic swelling with rapid volume restoration achieved by increased permeability to potassium, and to chloride, with consequent loss of cellular KC1 [10,14,16-321. The enhanced potassium flux shares some properties with that induced by A23187 including blockade by quinine. However, other properties of the two potassium permeabilities are rather different including the sensitivity to trifluoperazine and the 86Rb fluxes requirement for external calcium [26,31]. stimulated by A23187 are largely dependent on external calcium. The volume regulation does not require the presence of external calcium, although a prolonged incubation in calcium free medium inhibits the volume restoration. Recent studies with the calcium indicator quin2 show that hypotonic swelling did not alter [Ca2+]i and that volume restoration occurred while calcium remained unchanged at basal levels [15]. It seems therefore that the swellin increase in potassium -induced permeability is activated t y some means other than elevated [Ca2+]i. Further study will be needed to see if 467
the two conditions same channel or to potassium channel.
lead to a different activation activation of different types
of of
the
MITOGEN-INDUCEDALTERATION IN POTASSIUM PERMEABILITY There are numerous reports of increased cation fluxes following mitogenic stimulation of lymphocytes [2,9,13,33-431. The findings have varied according to the preparation and methods used, the amount and type of mitogen and the period of observation. In many cases an increased potassium flux has been observed. In the recent patch-clamp study there was a c. 2-fold enhancement of potassium currents in mitogen-stimulated human lymphocytes In some instances there is evidence that mitogens Ill. may stimulate a calcium-activated potassium conductance. The mitogenic lectin, concanavalin A, was found to cause an early elevation of [Ca2+1- and a concurrent hyperpolarization in mouse tAymocytes [13]. This response was dependent on external calcium and blocked by quinine but not by ouabain. Therefore the concanavalin A-induced response was consistent with activation of potassium channels by the elevated [Ca2+]i. A similar hyperpolarization of mouse thymocytes was observed by Felber and Brand [22], who also reported that concanavalin A increased 86Rb influx. In pig lymphocytes, however, concanavalin A raises [Ca 2+]i [13] but apparently dggibnot cause a measurable hyperpolarization, or increased As discussed in connection with the failure of flux [22]. calcium ionophore to hyperpolarize pig cells, this could be due to the presence of a fully activated calcium-activated potassium conductance, or to the absence Studies of mitogenic of such a conductance. transformation in human peripheral lymphocytes have given conflicting estimates of membrane potential changes; some authors reporting a depolarization [e.g. 211, others no In preliminary measurements significa;t change [e.g. 191. A produced no with quin , it was found that concanavalin measurable change in [Ca2+]i in human peripheral blood observation] in lymphocytes [Rink, T. J., unpublished contrast to the results with mouse thymocytes and pig mesenteric node lymphocytes. The significance of the hyperpolarization induced by One concanavalin A in thymocytes remains obscure. possible role for a hyperpolarization of the membrane is that this could help drive an increased sodium-linked uptake of organic substance such as glucose and amino have suggested that acids. However, two groups [19,221 membrane potential plays no critical role in the response to lectins at least in the first two to three hours of stimulation.
468
CONCLUSION The finding
calcium ionophore can produce a 86Rb flux in several lymphocyte preparations presently provides the strongest evidence for the existence of calcium-activated potassium channels. The presence of such channels is also consistent with the observations that (i) elevations of [Ca*+Ii by ionophore or mitogenic lectins can hyperpolarize some types of and (ii) that substances known to block these lymphocytes, channels in other cells can prevent this hyperpolarizaand can depolarize the resting membranes of some tion, It seems likely that there are several kinds lymphocytes. of potassium channel in lymphocyte membranes and that not all lymphocytes have the same membrane properties. It is expected that application of modern electrophysiological techniques particularly the patch-clamp in its various forms will soon allow a detailed analysis of the electrical properties of defined sub-populations of lymphocytes. This should resolve many questions about the existence, nature and functions of these channels. specific
increase
that in
ACKNOWLEDGEMENTS The authors ’ work has been supported by grants from the SERC, UK, and the NIH, U.S.A. C.D. holds a Research Career Development Award. We thank Dr. M. Brand and MS S. Felber for helpful discussion. REFERENCES 1.
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Received Accepted
3.10.83 2.11.83
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