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ION CHANNELS AND SIGNAL Annu. Rev. Physiol. 1990.52:415-430. Downloaded from Access provided by University of California - Davis on 01/26/15. For personal use only.

TRANSDUCTION IN LYMPHOCYTES Richard S. Lewis and Michael D. Cahalan Department of Physiology and Biophysics, California College of Medicine, University of California, Irvine, California 92717 KEY WORDS:

cell activation, volume regulation, ion transport, patch-clamp, calcium signaling

INTRODUCTION and B lymphocytes play fundamental and diverse roles in the immune response, which include recognition of antigens, secretion of Iymphokines and antibodies, and killing of foreign or virus-infected cells. Ionic fluxes associated with lymphocyte activation, mitogenesis, and volume regulation have been studied extensively in cell suspensions (for reviews, see 67, 103, 124, Grinstein & Foskett, this volume). More recently, the advent of patch­ clamp recording techniques (55) and video imaging of fluorescent ion­ sensitive dyes (53, 131) has placed a new emphasis on the responses of individual cells in the immune system. In this review we address recent advances in the exploration of ion-channel functions in T and B lymphocytes at the single-cell level, with particular emphasis on three lymphocyte be­ haviors: cell activation and mitogenesis, cytotoxicity, and volume regulation.


LYMPHOCYTE BEHAVIORS AND TRANSMEMBRANE ION FLUXES Lymphocytes are activated in vivo by the binding of specific antigens to the T-cell receptor complex (for T cells) or surface immunoglobulin molecules (for B cells). Monoclonal antibodies that cross-link selected cell surface glycoproteins, as well as several lectins, are commonly used together with 0066-4278/90/0315-0415$02.00


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phorbol esters to initiate cell activation in vitro. The ensuing activation response encompasses a coordinated series of lineage-dependent and -inde­ pendent differentiation events, culminating in DNA synthesis and cell divi­ sion (reviewed in 14, 81). Some of the earliest events, commencing within minutes of exposure to mitogens, include transmembrane ion movements, efflux of K+ (41, 121) and H+ (57, 108), and influx of Na+ (30, 101, 121) and Ca2+ (61, 132, 137, reviewed in 134). During the same time period membrane phosphatidy1inositol 4,5-bisphosphate is hydrolyzed, yielding ino­ sitol 1,4,5-trisphosphate (IP3) , which mobilizes Ca2+ from intracellular stores, and diacylglycerol, which activates protein kinase C (14, 134). Com­ mitment of lymphocytes to the activation pathway is incompletely un­ derstood, but it is generally thought to involve both a sustained rise of intracellular free Ca2+ concentration ([Ca2+]j) and activation of protein kinase C, based on several lines of evidence. First, many Ca2+-mobilizing stimuli, when combined with phorbol esters, activate T cells (134) , while preventing the sustained [Ca2+]j rise prevents cell activation (42, 56, 93, 135, 136). In addition, elevation of intracellular [Ca2+] with ionophores, in the presence of phorbol esters, bypasses the involvement of cell-surface receptors and activates lymphocytes (86, 89, 130, 133), leading to expression of early activation genes, such as c-fos, c-myc, and the T-cell-specific interleukin-2 (IL-2) gene (22, 94, 105, 134). Ca2+ -independent pathways may also exist, however, as a variant T-cell hybridoma can be activated by surface-receptor crosslinking to secr�te IL-2 in the absence of a detectable [Ca2+L rise (125). The process by which cytotoxic T cells and natural killer cells destroy foreign or virus-infected cells is thought to occur in three phases, referred to as binding, programming-for-Iysis, and lethal hit. Although binding of the effector cell to its target is Ca2+ -independent, programming and delivery of the lethal hit require extracellular Ca2+ (84). K+ efflux (109) and Ca2+ influx (100) in the effector cells both accompany and are required for the cell-killing reaction. When exposed to a hypotonic environment, T cells swell passively, then spontaneously shrink over a period of minutes to approximately their normal volume. This regulatory volume decrease (RVD) is characteristic of a variety of cell types and is reviewed in detail by Grinstein & Foskett in this volume. RVD is mediated through the efflux of K+ and Cl- from the cell, which drives out osmotically obligated water and causes the cell to shrink (18, 45, 112-114). The patch-clamp technique (55) has enabled characterization of a variety of ion channels in cells of the immune system, several of which appear to underlie the ion fluxes described above (for reviews, see 12, 24, 36). The high membrane resistance (10-100 G 0) and low surface area (1-2 x 10-6 cm2) and volume (2-3 X 10-10 cm3) of lymphocytes make it possible for small numbers of ion channels to have a powerful influence on both mem-



brane potential and intracellular [Ca2+]. For these reasons, the patch-clamp technique, offering both high sensitivity and the ability to introduce specific second messengers into the cytoplasm

(95), is ideally suited to the detailed

study of channel function and regulation in lymphocytes. The following sections review the present knowledge of lymphocyte K+, CI-, and Ca2+ channels as derived from patch-clamp experiments.

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Properties Three distinct types of voltage-gated K+ channels have been identified in lymphoid cells based on their voltage dependence, tendency to inactivate, opening and closing kinetics, and pharmacologic profiles (for review, see The most commonly observed K+ channel, named type normal human



for its prevalence in

T cells (11, 23, 28, 87), opens at potentials positive to



m V, a voltage dependence that enables the cell to maintain a negative membrane potential. Type


K + channels are activated by depolarization per

se, not by intracellular Ca2+; instead, raising [Ca2+]i accelerates inactivation (to,

21, 50). These channels have a unitary conductance of 12-16 pS and are

sensitive to a variety of classical K+ channel blockers, such as tetraethylam­ monium (TEA) and 4-aminopyridine (4-AP)

(23, 51). Interestingly, type n

channels are also blocked by drugs thought at one time to be specific inhibitors of Ca2+-activated K+ channels, including quinine (23, 87, 116) and charybdotoxin (CTX) (111); by classical Ca2+-channel blockers, such as verapamil, diltiazem, dihydropyridines, Ni2+, Cd2+, and Co2+ (15, 24, 87); and by the calmodulin antagonists chlorpromazine and trifluopera­ zine (24). Two additional varieties ofK+ channels have been characterized in murine T cells and thymocytes. Compared with type


K+ channels,


channels do

not accumulate inactivation during repetitive depolarizing pulses, and are =

to-fold less sensitive to block by TEA, although they display a similar

sensitivity to CTX and similar unitary conductance

(75). Type I K+ channels,

named for their larger conductance (21-27 pS) and prevalence in autoimmune mice bearing the lpr mutation, are even more distinct, showing a lack of cumulative inactivation, a more positive voltage threshold for activation, faster closing kinetics, and a to-fold higher sensitivity to block by TEA compared to n or CTX

' n


(16, 25, 75). Type I channels are not blocked by


Expression Subtypes of K+ channels are distributed in a characteristic pattern among functional and developmental subsets of T cells. Type


channels are the most

common and are found in human peripheral blood T cells

(23, 87), thymo-

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cytes (117), and leukemic cell lines (24), and in most subsets of murine thymocytes (immature CD4-CDS- and CD4+CDS+ cells, and helper­ phenotype CD4+CDS- cells; 75, 90), peripheral T cells, and clonal cell lines (24, 25, 34, 74). Type n channels have also been detected in B lymphocytes (127), macrophages (38, 102, 141), platelets (85), and type II alveolar epithelial cells (27). In contrast, expression of type n ' and I channels is restricted to murine thymocytes and peripheral T cells of the cytotoxic/ suppressor phenotype (CD4.-CDS+) (52, 75), while abundant expression of type I channels marks functionally aberrant CD4-CDS- T cells from mice with autoimmune disease (16, 52). Thus far, type n ' or I channels have not been observed in human T or B cells, although I channels are present in a human B-cell lymphoma line (122).

Functions Several lines of evidence point to a requirement for functional n-type K+ channels in the activation of T and B lymphocytes by mitogens. First, expression of type n K+ channels is correlated with the degree of cell proliferation. In the thymus, actively cycling cell subsets express 10- to 20-fold the K+-channel density of quiescent cells (75, 90), and K+ channel expression in resting peripheral T and B cells is boosted by a similar amount following mitogenic stimulation (26, 74, 126, 127). In addition, mitogenic lectins have been reported to affect the voltage dependence of K+ channel gating (11, 23, 24, 117) and maximum K+ conductance (117) of thymocytes and T cells, but the mechanism of these acute effects and their physiologic significance remain to be determined. The most compelling evidence for a role of K+ channels in lymphocyte activation is the dose-dependent inhibition of mitogenesis by K+ channel blocking agents. A variety of structurally distinct compounds, including TEA, 4-AP, quinine, cetiedil, verapamil, nifedipine, and diltiazem, inhibit mitogenesis of T and B cells and antibody secretion by B cells at levels required to block type n channels (15, 16, 23, 24, 2S, 110, 115, 126, 127). K+ channels appear to be necessary for some, but not all, cell activation events (15, 110); for example, while protein and DNA synthesis and IL-2 production are inhibited by the blockers, expression of IL-2 receptors is unaffected. K+ channel blockers also inhibit the cytotoxic activity of human natural killer cells, apparently by interfering with the programming phase of the reaction (116, 123). Similar effects have been observed using human cytotoxic T cells and a murine natural killer cell line (K. G. Chandy, et aI, unpublished observations). The important question remains, how do K+ channels exert their influence on lymphocyte activation? The sensitivity of mitogenesis to K+ channel blockade declines after"several hours of mitogen stimulation, which suggests a role during early stages of cell-cycle progression (15, 110). One hypothesis,

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that K+ channels conduct Ca2+ into the cell (11), now appears unlikely, as full K+ channel activation by voltage-clamp depolarization does not alter [Ca2+]j measured in single T cells with fura-2 (78). K+ channel blockers depolarize T cells (138), and depolarization by high [K+]o inhibits ca2+ signaling (40, 43, 97), thereby supporting the notion that K+ channels indirectly facilitate Ca2+ influx through mitogen-stimulated Ca2+ channels by maintaining a negative membrane potential (11). However, K+ channel blockers, such as TEA and 4-AP, only reduce the early [Ca2+]j rise evoked by phytohemagglutinin (PHA) in T cells by 10-25% (41). This issue may be resolved when the blockers are tested for longer-term effects on [Ca2+]j. Functional K+ channels may be required for normal transport of essential metabolites, since TEA and 4-AP have been found to inhibit uptake of thymidine and phenylalanine by cloned T cells (115). These inhibitory effects are most likely the result of K+ channel block rather than of nonspecific action of the blockers themselves, since these agents do not inhibit growth of a lymphoid cell line known to lack K+ channels (15). Two types of experimental evidence suggest that type n K+ channels are involved in the RVD response of T lymphocytes. First, the abundance of n channels directly parallels the ability of T and B cells to volume regulate (19, 28, 73, 127). Second, drugs known to block type n channels in T cells, such as TEA, quinine, cetiedil, verapamil, trifluoperazine, chlorpromazine, and CTX, inhibit RVD at comparable concentrations (13, 18, 24, 47, 48, 73, 113). RVD and type n K+ channels also exhibit similar sensitivities to intracellular pH (28a). It has been proposed that Cl- channels activated by osmotic swelling depolarize the membrane to open n-type K+ channels during RVD (13). CALCIUM-ACTIVATED POTASSIUM CHANNELS At least two major classes of Ca2+-activated K+ channels have been found in the nervous system: a high-conductance (=200 pS) maxi-K+ channel that is opened by depolarization and intracellular Ca2+ and is highly sensitive to block by the scorpion toxin component, CTX (2, 3, 92); and a lower­ conductance, voltage-insensitive, Ca2+ -activated channel that is blocked by the bee venom peptide, apamin (8, 60). Indirect evidence supporting the existence of Ca2+ -activated K+ channels in lymphocytes includes membrane hyperpolarization and 86Rb + flux induced by raising rCa2+]j with ionophores or mitogens (29, 46, 47, 82, 128, 132, 138). The early work on this subject has been reviewed (106). Two recent patch-clamp studies have identified Ca2+ -activated K+ chan­ nels in rat thymocytes and human B cells (83a) and in lurkat leukemic T cells (49). In cell-attached patches exposed to 140 mM K+, K+ -selective channels

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with conductances of 8 and 25 pS are activated upon elevation of [Ca2+]j (83a). The channels show little voltage dependence and in excised patches exhibit a threshold of several hundred nanomolar Ca2+ for activation. Gallin (37) has described a similar 36-pS K+ channel activated in human mac­ rophages by the Ca2+ ionophore, ionomycin. The time course of Ca2+­ activated K+ current in single Jurkat T cells follows the transient elevation of [Ca2+]j induced by ionomycin or PHA (49; R. S. Lewis & M. D. Cahalan, unpublished observations), and the current is blocked by apamin (S. Grissmer & M.D. Cahalan, unpublished observations). Ca2+ -activated K+ channels are likely to be involved in generating the membrane hyperpolarization observed in mitogen-treated B and T cells, since the time-course of the hyperpolarization closely parallels that of the [Ca2+]j increase (82, 132). By maintaining a negative resting potential these channels may enhance the driving force for Ca2+ influx. Until recently, no ligand specific for the Ca2+ -activated K+ channel in lymphocytes was known (quinine and CTX block voltage-gated K+ channels in lymphocytes). The ability to selectively block Ca2+ -activated K+ channels with apamin should allow tests of their involvement in T- and B-cell activation. The possibility that Ca2+ -activated K+ channels constitute the K+ efflux pathway of RVD was proposed originally by Grinstein & colleagues based on observations that RVD is inhibited by quinine, calmodulin antagonists, or prolonged incubation in Ca2+-free medium, and that calcium ionophores can induce cell shrinkage (46, 47). Such a role is questionable, however, as a [Ca2+]j rise is undetectable during RVD of human T cells (107), and the pharmacologic sensitivity of K+ efflux during RVD closely parallels that of voltage-gated, type n K+ channels (discussed above). CHLORIDE CHANNELS Several varieties of Cl- channels have been found in lymphocytes, which cover a spectrum of unitary conductances: a 1-2-pS mini channel (13), a 40-pS channel (17), and a 300--400-pS maxi-CI- channel (12, 120). Because the normal lymphocyte resting potential of -60 mV lies below the estimated Cl- equilibrium potential of =-35 mY, activation of Cl- channels causes Cl- efflux and membrane depolarization (45). Mini-CI- channels have been found in human and murine T cells and related lines and in both immature and mature thymocyte subsets in mice (13; R. S. Lewis & M. D. Cahalan, unpublished observations). Single channels are too small to be clearly evident in whole-cell recording; the unitary conductance is estimated from fluctuation analysis to be 1-2 pS. Cl- channel activation is triggered during whole-cell recording by hypoosmotic ex-

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tracellular or hyperosmotic intracellular solutions and requires the presence of intracellular ATP. Suction applied to the whole-cell recording pipette retards or reverses the activation of the current induced by hypotonic media, thus suggesting that these channels are activated by membrane stretch produced by cell swelling. The acute modulation of mini-CI- channels by changes in extracellular tonicity suggests that they underlie the anion transport limb of the RVD pathway. This conclusion is supported by the parallel anion selectiv­ ity of the Cl- conductance (N03" > Br- ?;: Cl -) and of RVD (13, 45). Identification of blocking compounds for the mini-CI- channels wi ll facilitate further tests of their role in RVD. A second type of CI- channel has been described recently in human leukemic T cells and transformed B lymphoblasts (17). Its unitary con­ ductance of 40 pS and its activation by prolonged membrane depolarization or by intracellular cAMP and cAMP-dependent protein kinase suggest a close resemblance to the Cl- channels of secretory cells in airway epithelia (80, 119). The role of the 40-pS Cl- channel in lymphocyte functions is not known. Regulation of the channels by CAMP-dependent protein kinase, however, appears to be defective in lymphoblasts derived from patients with cystic fibrosis (CF) (17). A similar defect is known to affect Cl- channels in airway epithelia from CF patients and underlies the pathogenesis of the disease (80, 119). The finding that lymphocytes, a highly accessible biologi­ cal material, may express the same channel-regulation defect suggests their use as a model preparation for both diagnosis and research on the molecular basis of defective Cl- transport in cystic fibrosis. Large conductance maxi-Cl- channels have been found in murine thymo­ cytes, T cells, and in a B-cell hybridoma (9, 12, 13, 120), as well as in a variety of other cell types (7, 44, 54, 69). The channels are easily recognized by their 300-400-pS conductance, multiple subconductance states, and volt­ age dependence. Activation is complex; the channels can be opened by several min of membrane depolarization to potentials of >0 mV, by treatment of the cell with ionomycin, or by patch excision. Once induced, the channel is maximally active at 0 mY, closing at both positive and negative potentials. The activation by ionomycin may involve Ca2+ -dependent intracellular biochemical events, since following patch excision the channel's activity is unaffected by changes in [Ca2+] bathing the intracellular face (120). A clear function for the maxi-CI- channel has not yet been demonstrated. In a recent report showing a similar channel in sarcoplasmic reticulum (SR) from skeletal muscle, Hals et al (54) propose that the channel may indirectly regulate Ca2+ release from the SR. If the maxi-CI channels also exist in the endoplasmic reticulum membranes of lymphocytes, one could envision an analogous role in regulating intracellular Ca2+ release during lymphocyte activation. �



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MITOGEN-ACTIVATED CALCIUM CHANNELS The existence of mitogen-regulated Ca2+ channels in T and B lymphocytes was initially inferred from indirect measurements, beginning with observa­ tions of 45Ca2+ uptake induced by polyclonal mitogens (32, 136, 137). Although Ca2+ influx is electrogenic (83), voltage-gated Ca2+ channels do not appear to be involved, since depolarization of cells with high [K+]o before or after exposure to mitogens does not elicit a rise in [Ca2+]i (40, 77, 97). Likewise, voltage-clamp depolarization fails to evoke inward Ca2+ current in Tor B cells (11, 34, 126), although voltage-gated Ca2+ channels are detect­ able in murine myeloma and B-cell hybridomas (33, 35). A recent report suggesting the presence of voltage-sensitive Ca2+ channels in lurkat leukemic T cells (28b) must be regarded with skepticism until the ability of Ca2+ to carry the current is established. Inhibition of 45Ca2+ uptake and mitogenesis by Ca2+ -channel blockers such as verapamil and dihydropyridines has been taken as evidence for mitogen-gated Ca2+ channels in T cells (6), although this interpretation is complicated by the fact that these compounds block type n K+ channels (15, 24). Three groups have reported direct measurements of mitogen-stimulated Ca2+ or Ba2+ currents in T cells or T-cell membranes, but no common consensus has been reached on their characteristics. Gardner & colleagues have described single-channel and whole-cell currents evoked in human T cells by PHA and antibodies to CD2 or CD3 (39, 70, 71). In cell-attached patches exposed to isotonic external BaClz, the channels have a conductance of =7 pS and show some selectivity for BaH. Channel openings are brief (0.4-0.5 msec), occur in short bursts, and are not measurably voltage­ dependent. The ability of bath-applied PHA to elicit channel activity in cell-attached patches indicates that the underlying activation mechanism in­ volves a diffusible intracellular second messenger. A possible role for 1, 4, 5IP3 in this regard is consistent with its ability to activate the channel in excised inside-out patches (70). Micromolar levels of intracellular Ca2+ block the channel reversibly, suggesting a means of auto-regulation during the mitogen­ ic response. Whole-cell currents induced by mitogens were large (up to -130 pA at -50 m V) and showed poor selectivity for BaH over monovalent cations (39, 71). A different type of antibody triggered channel activity has been described in bilayers formed from plasma membranes of a human T-cell line (98). Antibodies to CD3 or T-cell receptor epitopes unique to these cells elicit single-channel currents having a small conductance (2-6 pS in symmetrical 100 mM CaCI , 7-8 pS in symmetrical 50 mM BaCI ). These channels are 2 2 distinct from those described by Gardner & coworkers in several ways. First, channel opening persists in the presence of millimolar levels of intracellular

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Ca2+. Second, the openings are long-lived, in some instances lasting more than 50 sec. Finally, the frequency of openings decreases with depolarization. In addition, channel activation occurs in the bilayer presumably by direct interaction of the antibodies with the channel or an associated protein; in the experiments of Kuno et al (39, 70, 71), second messengers appear to be involved, although additional activation by direct interaction cannot be ruled out. Recently, a mitogen-activated Ca2+ current in T cells has been described using a combination of whole-cell or perforated-patch recording (58) and single cell Ca2+ measurement using fura-2 (77, 79). The advantage of this combined approach is that it allows the correlation of particular components of membrane current with the [Ca2+]i rise in an individual cell. This advan­ tage is an important one because, given the cell volume of lymphocytes, an extremely small Ca2+ -selective inward current in principle could suffice to elevate [Ca2+]i to micromolar levels. PHA treatment of Jurkat cells induces a small inward Ca2+ current « 1 to �1O pA) after a delay of 100--300 sec, closely followed by a rise in [Ca2+]i. The conductance is not voltage­ dependent and appears to be inhibited by intracellular Ca2+ (77). In some cases the current undergoes periodic slow fluctuations that closely parallel [Ca2+]i oscillations (see below), which is consistent with a role in the oscillation mechanism. The high selectivity of the current for Ca2+ distin­ guishes it from the mitogen-gated, whole-cell currents reported previously (71). Its precise relationship to the single-channel events reported by Gardner & colleagues (39, 70, 71) is at present unclear. The absence of readily detectable current fluctuations during its induction suggests that, if the current is conducted by channels, they must have an extremely small conductance or exceedingly brief openings (R. S. Lewis & M. D. Cahalan, unpublished observations). A similarly smooth Ca2+ current has been reported in mast cells following stimulation by substance P or compound 48/80 and is believed to represent the major Ca2+ influx pathway activated by those agents (88, 99). Transmembrane Ca2+ flux can be evoked in T and B lymphocytes by a variety of stimuli. For T cells, Ca2+ signals have been elicited by antigenic recognition, mitogenic lectins, and cross-linking antibodies directed against the cell-surface glycoproteins CD2 through CD8, CD28, thy-I , and the T cell receptor (1, 31, 59, 62, 63, 65, 66, 68, 72, 96, 129, 133). For B cells, the most effective stimulus is anti-Ig antibodies (5, 83, 104, 139). For at least several of these stimuli, the Ca2+ rise occurs via release of Ca2+ from intracellular pools, with influx being necessary to sustain the response (1, 5, 57, 61, 62, 65, 77, 79, 104). Several kinds of indirect evidence, reviewed above, suggest that a sustained [Ca2+]i rise is an important triggering signal for lymphocyte activation. The identification of blocking drugs specific for the mitogen-regulated Ca2+ channel should greatly facilitate future studies of -

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its role in transducing extracellular stimuli into cytosolic Ca2+ signals for lymphocyte activation and other behaviors such as cytotoxicity. An unsuspected degree of complexity in the pattern of mitogen-stimulated Ca2+ signaling in single cells has been revealed recently by dynamic measure­ ments of [Ca2+]j using fura-2. These experiments show that the sustained phase of the [Ca2+]j rise in T and B cells results largely from repetitive, asynchronous [Ca2+]j oscillations occurring at the level of individual cells (77, 79, 131, 139). Oscillations of [Ca2+]j are a common consequence of cell activation in a number of systems (4, 64, 131, 140). There is currently great interest in identifying the molecular basis of the oscillation mechanism, and several models have been proposed that involve repetitive release from in­ tracellular stores (4, 4a, 91) or oscillatory Ca2+ influx across the plasma membrane (77).

CONCLUSIONS AND FUTURE DIRECTIONS Patch-clamp techniques have enabled the characterization of a diverse set of ion channels in lymphocytes with diverse functional roles. One theme guiding future work in this area is the control of ion-channel activity by intracellular messengers. Regulated patterns of electrical activity in lymphocytes, includ­ ing the activation and deactivation of CI- channels during the volume regula­ tion response and the periodic activity of Ca2+ channels underlying mitogen­ triggered [Ca2+]j oscillations, most likely arise from biochemical or mechani­ cal rather than electrical feedback because the underlying channels are vol­ tage-insensitive. Evidence exists for biochemical modulation of voltage­ dependent n-type K+ channels by cAMP and serotonin in B cells (20, 21), which suggests that neurotransmitters may influence immune function through their effects on ion channels. The Single-cell approach to studying membrane properties is a powerful one. In addition to providing high-resolution detection of ion channels down to the level of single-channel molecules in the cell, advantages include the ability to characterize properties of identified sUbpopulations of cells (as in thymocytes), to reveal dynamic aspects of cell behavior that are obscured in cell suspensions (e.g. [Ca2+]j oscillations), and to temporally correlate ion channel activity with ionic signals. An increasingly effective and sophisti­ cated array of experimental approaches is being developed to study ionic signaling and modulation at the single -cell level that includes perforated­ patch recording, photoreleasable messengers, and imaging with ion-sensitive dyes. As imaging techniques evolve to provide for real-time detection of enzyme activity and gene transcription, the entire signal transduction pathway from membrane to nucleus may ultimately be visualized in single cells.




The authors' work was supported by National Institutes of Health grants GM41514 and NS14609, and the Office of Naval Research.

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