Microglial SK3 and SK4 currents and activation state are modulated by the neuroprotective drug, riluzole.

Microglia monitor the CNS for 'danger' signals after acute injury, such as stroke and trauma, and then undergo complex activation processes. Classical activation of microglia can produce neurotoxic levels of glutamate and immune mediators (e.g., pro-inflammatory cytokines, reactive oxygen and nitrogen species), while alternative activation up-regulates anti-inflammatory molecules and is thought to resolve inflammation and protect the brain. Thus, pharmacological strategies to decrease classical- and/or promote alternative activation are of interest. Here, we assessed actions of the neuroprotective drug, riluzole, on two Ca(2+)- activated K channels in microglia - SK3 (KCa2.3, KCNN3) and SK4 (KCa3.1, KCNN4) - and on classical versus alternative microglial activation. Riluzole is used to treat amyotrophic lateral sclerosis, and is in clinical trials for several other CNS disorders, where it has been presumed to target neurons and reduce glutamate-mediated toxicity. We show that simply elevating intracellular Ca(2+) to micromolar levels in whole-cell recordings does not activate SK channels in a cell line derived from primary rat microglia (MLS-9). In intact cells, riluzole raised cytoplasmic Ca(2+), but it was marginal (~200 nM) and transient (2 min). Surprisingly then, in whole cell recordings, riluzole rapidly activated SK3 and SK4 channels for as long as it was present, and did not require elevated intracellular Ca(2+). We then used primary rat microglia to analyze expression of several activation markers and inflammatory mediators. Riluzole decreased classical LPS-induced activation, and increased some aspects of IL-4-induced alternative activation. These actions on microglia suggest an additional mechanism underlying the neuroprotective actions of riluzole.

The repulsive guidance molecule, RGMa, promotes retinal ganglion cell survival in vitro and in vivo.

The repulsive guidance molecule, RGMa, and its receptor Neogenin, regulate neuronal cell death during development, but little is known about their expression and roles in the adult CNS. Here, we show that Neogenin is expressed in the adult rodent retina, particularly on retinal ganglion cells. To determine whether the Neogenin/RGMa pathway is important in the fully developed retina, we examined its contribution to damage-induced neurodegeneration. The effects of RGMa on survival of retinal ganglion cells (RGCs) were examined in vitro and in vivo. Using cultured whole-mount retinal explants, we showed that the addition of RGMa increased RGC survival and that this effect was mediated by the Neogenin receptor. Immunohistochemical analysis indicated that the inhibition of cell death by RGMa resulted from reduced caspase-3 activation. Then, using an in vivo model of RGC apoptosis after optic nerve transection, we demonstrated that intraocular injection of RGMa at 3 and 7 days after axotomy greatly reduced RGC death 14 days postaxotomy. This study provides the first evidence that RGMa is a molecular target for neuroprotection in retinal pathologies, and suggests that targeting "dependence receptors" such as Neogenin has therapeutic potential for the treatment of neuropathologies in the adult CNS.

Kv1.1 and Kv1.3 channels contribute to the degeneration of retinal ganglion cells after optic nerve transection in vivo.

Degeneration of retinal ganglion cells (RGCs) - an important cause of visual impairment - is often modeled by optic nerve transection, which leads to apoptotic death of these central nervous system neurons. With this model, we show that specific voltage-gated K(+) channels (Kv1 family) contribute to the degeneration of rat RGCs and expression of apoptosis-related molecules in vivo. Retinal expression of Kv1.1, Kv1.2, Kv1.3 and Kv1.5 was examined by quantitative real-time reverse transcriptase-PCR and immunohistochemistry. Kv channel blockers and channel-specific short-interfering RNAs (siRNAs) were used to assess their roles in RGC degeneration. We found that (i) rat RGCs express Kv1.1, Kv1.2 and Kv1.3 (but not Kv1.5); (ii) intraocular injection of agitoxin-2 or margatoxin, potent blockers of Kv1.1, Kv1.2 and Kv1.3 channels, dose-dependently reduced the RGC degeneration; (iii) siRNAs applied to the cut optic nerve were rapidly transported throughout RGCs only, in which they reduced the expression of the cognate channel only. Our results show differential roles of the channels; siRNAs directed against Kv1.1 or Kv1.3 channels greatly reduced RGC death, whereas Kv1.2-targeted siRNAs had only a small effect, and siRNAs against Kv1.5 were without effect. (iv) Kv1.1 and Kv1.3 channels apparently contribute to cell-autonomous death of RGCs through different components of the apoptotic machinery. Kv1.1 depletion increased the antiapoptotic gene, Bcl-X(L), whereas Kv1.3 depletion reduced the proapoptotic genes, caspase-3, caspase-9 and Bad.

Association between cerebrospinal fluid interleukin-6 concentrations and outcome after severe human traumatic brain injury.

Acute inflammation plays a significant role in the pathophysiology of traumatic brain injury (TBI). However, the specific relationships between inflammatory mediators and patient outcome following TBI have not been fully established. In this study, we measured plasma and cerebrospinal fluid interleukin-1 (IL-1) and interleukin-6 (IL-6) concentrations in 36 patients, following severe TBI. Patients were monitored with continuous measurements of somatosensory-evoked potentials (SSEP) to derive an established surrogate outcome measurement, the 96-h evoked potential (SSEP96). Clinical outcomes were assessed at 3 months using the Glasgow Outcome Scale (GOS). Peak cerebrospinal fluid (CSF) IL-1 and IL-6 concentrations were significantly higher than those observed in the plasma [median 6.5 pg/mL (range 1.4-25.0) vs. 3.0 (0.8-7.6) for IL-1, and 650 (130-7,214) vs. 253 (52-1,506) for IL-6, p < 0.001 for both]. Peak CSF IL-6 levels correlated with SSEP96 (r = 0.42; p = 0.0133), and peak CSF IL-6 levels were higher with improved GOS (p = 0.024). Multiple regression analysis identified that age (p = 0.0072), pupillary abnormality (p = 0.021), the presence of mass lesion (p = 0.023), and peak CSF IL-6 concentrations (p = 0.026) were all statistically significant predictors of clinical outcome following TBI. These results suggest that peak CSF IL-6 concentrations correlate with improved outcome following TBI. This finding helps to characterize the inflammatory reaction associated with TBI and may help to develop improved treatment strategies for patients with TBI.

Calmodulin regulates assembly and trafficking of SK4/IK1 Ca2+-activated K+ channels.

Calmodulin (CaM) regulates gating of several types of ion channels but has not been implicated in channel assembly or trafficking. For the SK4/IK1 K+ channel, CaM bound to the proximal C terminus ("Ct1 " domain) acts as the Ca2+ sensor. We now show that CaM interacting with the C terminus of SK4 also controls channel assembly and surface expression. In transfected cells, removing free CaM by overexpressing the CaM-binding domain, Ct1, redistributed full-length SK4 protein from the plasma membrane to the cytoplasm and decreased whole-cell currents. Making more CaM protein available by overexpressing the CaM gene abrogated the dominant-negative effect of Ct1 and restored both surface expression of SK4 protein and whole-cell currents. The distal C-terminal domain ("Ct2") also plays a role in assembly, but is not CaM-dependent. Co-immunoprecipitation experiments demonstrated that multimerization of SK4 subunits was enhanced by CaM and inhibited by removal of CaM, indicating that CaM regulates trafficking of SK4 by affecting the assembly of channels. Our results support a model in which CaM-dependent association of SK4 monomers at their Ct1 domains regulates channel assembly and surface expression. This appears to represent a novel mechanism for controlling ion channels, and consequently, the cellular functions that depend on them.

Regulation of Kv1.3 channels in activated human T lymphocytes by Ca(2+)-dependent pathways.

Activated T lymphoblasts respond more effectively to mitogenic stimuli than resting T cells, partly through differences in Ca(2+) signaling, which in turn depend on K(+) channel activity. Both Kv1.3 and Ca(2+)-activated K(+) (SK4) channels are up-regulated in T lymphoblasts. Since Ca(2+)- and calmodulin (CaM)-dependent signal-ing are key pathways in T-cell activation, we investigated their involvement in regulating the Kv1.3 current. Kv1.3 in lymphoblasts was significantly inhibited by elevating internal Ca(2+) to the micromolar level. It was also reduced in a Ca(2+)-dependent manner by inhibiting CaM with W-7 or calmidazolium. Part of the CaM-dependence is likely through CaM kinase since the current was also inhibited by the antagonist, KN-62, but not by the inactive analogue, KN-04. Kinase inhibition, unlike CaM inhibition, was only effective at physiological temperatures, a difference that implies involvement of more than one mechanism. We demonstrated a biochemical association of Kv1.3 protein in lymphoblasts with the multifunctional type II CaM kinase, but not with calmodulin. Thus, Kv1.3 forms a multi-protein complex with CaM kinase II (which binds to Ca(2+)/CaM) and previously identified proteins (e.g., PSD-95, src tyrosine kinase) that position the channel to respond to signaling pathways that are crucial for T-cell activation and proliferation.

K+ channels and the microglial respiratory burst.

Microglial activation following central nervous system damage or disease often culminates in a respiratory burst that is necessary for antimicrobial function, but, paradoxically, can damage bystander cells. We show that several K+ channels are expressed and play a role in the respiratory burst of cultured rat microglia. Three pharmacologically separable K+ currents had properties of Kv1.3 and the Ca2+/calmodulin-gated channels, SK2, SK3, and SK4. mRNA was detected for Kv1.3, Kv1.5, SK2, and/or SK3, and SK4. Protein was detected for Kv1.3, Kv1.5, and SK3 (selective SK2 and SK4 antibodies not available). No Kv1.5-like current was detected, and confocal immunofluorescence showed the protein to be subcellular, in contrast to the robust membrane localization of Kv1.3. To determine whether any of these channels play a role in microglial activation, a respiratory burst was stimulated with phorbol 12-myristate 13-acetate and measured using a single cell, fluorescence-based dihydrorhodamine 123 assay. The respiratory burst was markedly inhibited by blockers of SK2 (apamin) and SK4 channels (clotrimazole and charybdotoxin), and to a lesser extent, by the potent Kv1.3 blocker agitoxin-2.

Suppression of the rat microglia Kv1.3 current by src-family tyrosine kinases and oxygen/glucose deprivation.

Microglia activate following numerous acute insults to the brain, including oxygen/glucose deprivation (OGD), and both protein tyrosine kinases (PTKs) and K+ channels have been implicated in their activation. We identified Kv1.3 (voltage-gated potassium channel) protein in cultured rat microglia and confirmed that the native current is biophysically and pharmacologically similar to Kv1. 3. To explore whether src-family PTKs regulate the microglial Kv current, we first heterologously expressed Kv1.3 in a microglia-like cell line derived from neonatal rat brain (MLS-9). The resulting large Kv1.3 current was eliminated by co-transfecting the constitutively active PTK, v-src, then rapidly restored by the PTK inhibitor, lavendustin A. Acute activation of endogenous src kinases by a peptide activator significantly reduced the current, an effect that was mimicked by OGD. Similarly, in primary cultures of rat microglia, the endogenous Kv1.3-like current was inhibited by activating endogenous src-family PTKs and by OGD. Biochemical analysis showed that OGD increased the tyrosine phosphorylation of native Kv1.3 protein, which was alleviated by PTK inhibitors or reactive oxygen species (ROS) scavengers. Conversely, the basal level of Kv1.3 phosphorylation was decreased by PTK inhibitors or scavengers of ROS. Together, our results point to a post-insertional downregulation of the microglial Kv1.3-like current by oxidative stress and tyrosine phosphorylation. This interaction may be facilitated by a multiprotein complex because, in cultured microglia, the endogenous Kv1.3 and src proteins both bind to the scaffolding protein, post-synaptic density protein 95 (PSD-95). By associating with, and phosphorylating Kv1.3, src is well positioned to regulate microglial responses to oxidative stress.

Internalization of the Kv1.4 potassium channel is suppressed by clustering interactions with PSD-95.

The contribution of voltage-dependent ion channels to nerve function depends upon their cell-surface distributions. Nevertheless, the mechanisms underlying channel localization are poorly understood. Two phenomena appear particularly important: the clustering of channels by membrane-associated guanylate kinases (MAGUKs), such as PSD-95, and the regional stabilization of cell-surface proteins by differential suppression of endocytosis. Could these phenomena be related? To test this possibility we examined the effect of PSD-95 on the internalization rate of Kv1.4 K(+) channels in transfected HEK293 cells using cell-surface biotinylation assays. When expressed alone Kv1.4 was internalized with a half-life of 87 min, but, in the presence of PSD-95, Kv1.4 internalization was completely suppressed. Immunochemistry and electrophysiology showed PSD-95 had little effect on total or cell-surface levels of Kv1.4 or on current amplitude, activation, or inactivation kinetics. Clustering was necessary and sufficient to suppress Kv1.4 internalization since C35S-PSD-95, a mutant reported to bind but not cluster Kv1.4, (confirmed by imaging cells co-expressing a functional, GFP-variant-tagged Kv1.4) restored and, surprisingly, enhanced the rate of Kv1.4 internalization (t((1)/(2)) = 16 min). These data argue PSD-95-mediated clustering suppresses Kv1.4 internalization and suggest a fundamentally new role for PSD-95, and perhaps other MAGUKs, orchestrating the stabilization of channels at the cell-surface.

A Kv1.5 to Kv1.3 switch in endogenous hippocampal microglia and a role in proliferation.

The proliferation of microglia is a normal process in CNS development and in the defense against pathological insults, although, paradoxically, it contributes to several brain diseases. We have examined the types of voltage-activated K(+) currents (Kv) and their roles in microglial proliferation. Microglia were tissue-printed directly from the hippocampal region using brain slices from 5- to 14-d-old rats. Immediately after tissue prints were prepared, unipolar and bipolar microglia expressed a large Kv current, and the cells were not proliferating. Surprisingly, this current was biophysically and pharmacologically distinct from Kv1.3, which has been found in dissociated, cultured microglia, but it was very similar to Kv1.5. After several days in culture the microglia became highly proliferative, and although the Kv prevalence and current density decreased, many cells exhibited a prominent Kv that was indistinguishable from Kv1.3. The Kv1.5-like current was present in nonproliferating cells, whereas proliferating cells expressed the Kv1.3-like current. Immunocytochemical staining showed a dramatic shift in expression and localization of Kv1.3 and Kv1.5 proteins in microglia: Kv1.5 moving away from the surface and Kv1.3 moving to the surface as the cells were cultured. K(+) channel blockers inhibited proliferation, and the pharmacology of this inhibition correlated with the type of Kv current expressed. Our study, which introduces a method for the physiological examination of microglia from identified brain regions, demonstrates the differential expression of two functional Kv subunits and shows that a functional delayed rectifier current is necessary for microglia proliferation.

Single-channel properties of BK-type calcium-activated potassium channels at a cholinergic presynaptic nerve terminal.

1. A high-conductance calcium-activated potassium channel (BK KCa) was characterized at a cholinergic presynaptic nerve terminal using the calyx synapse isolated from the chick ciliary ganglion. 2. The channel had a conductance of 210 pS in a 150 mM:150 mM K+ gradient, was highly selective for K+ over Na+, and was sensitive to block by external charybdotoxin or tetraethylammonium (TEA) and by internal Ba2+. At +60 mV it was activated by cytoplasmic calcium [Ca2+]i with a Kd of approximately 0.5 microM and a Hill coefficient of approximately 2.0. At 10 microM [Ca2+]i the channel was 50 % activated (V) at -8.0 mV with a voltage dependence (Boltzmann slope-factor) of 32.7 mV. The V values hyperpolarized with an increase in [Ca2+]i while the slope factors decreased. There were no overt differences in conductance or [Ca2+]i sensitivity between BK channels from the transmitter release face and the non-release face. 3. Open and closed times were fitted by two and three exponentials, respectively. The slow time constants were strongly affected by both [Ca2+]i and membrane potential changes. 4. In cell-attached patch recordings BK channel opening was enhanced by a prepulse permissive for calcium influx through the patch, suggesting that the channel can be activated by calcium ion influx through neighbouring calcium channels. 5. The properties of the presynaptic BK channel are well suited for rapid activation during the presynaptic depolarization and Ca2+ influx that are associated with transmitter release. This channel may play an important role in terminating release by rapid repolarization of the action potential.

hSK4/hIK1, a calmodulin-binding KCa channel in human T lymphocytes. Roles in proliferation and volume regulation.

Human T lymphocytes express a Ca2+-activated K+ current (IK), whose roles and regulation are poorly understood. We amplified hSK4 cDNA from human T lymphoblasts, and we showed that its biophysical and pharmacological properties when stably expressed in Chinese hamster ovary cells were essentially identical to the native IK current. In activated lymphoblasts, hSK4 mRNA increased 14.6-fold (Kv1.3 mRNA increased 1.3-fold), with functional consequences. Proliferation was inhibited when Kv1.3 and IK were blocked in naive T cells, but IK block alone inhibited re-stimulated lymphoblasts. IK and Kv1.3 were involved in volume regulation, but IK was more important, particularly in lymphoblasts. hSK4 lacks known Ca2+-binding sites; however, we mapped a Ca2+-dependent calmodulin (CaM)-binding site to the proximal C terminus (Ct1) of hSK4. Full-length hSK4 produced a highly negative membrane potential (Vm) in Chinese hamster ovary cells, whereas the channels did not function when either Ct1 or the distal C terminus was deleted (Vm approximately 0 mV). Native IK (but not expressed hSK4) current was inhibited by CaM and CaM kinase antagonists at physiological Vm values, suggesting modulation by an accessory molecule in native cells. Our results provide evidence for increased roles for IK/hSK4 in activated T cell functions; thus hSK4 may be a promising therapeutic target for disorders involving the secondary immune response.

HERG-like K+ channels in microglia.

A voltage-gated K+ conductance resembling that of the human ether-à-go-go-related gene product (HERG) was studied using whole-cell voltage-clamp recording, and found to be the predominant conductance at hyperpolarized potentials in a cell line (MLS-9) derived from primary cultures of rat microglia. Its behavior differed markedly from the classical inward rectifier K+ currents described previously in microglia, but closely resembled HERG currents in cardiac muscle and neuronal tissue. The HERG-like channels opened rapidly on hyperpolarization from 0 mV, and then decayed slowly into an absorbing closed state. The peak K+ conductance-voltage relation was half maximal at -59 mV with a slope factor of 18.6 mV. Availability, assessed by a hyperpolarizing test pulse from different holding potentials, was more steeply voltage dependent, and the midpoint was more positive (-14 vs. -39 mV) when determined by making the holding potential progressively more positive than more negative. The origin of this hysteresis is explored in a companion paper (Pennefather, P.S., W. Zhou, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:795-805). The pharmacological profile of the current differed from classical inward rectifier but closely resembled HERG. Block by Cs+ or Ba2+ occurred only at millimolar concentrations, La3+ blocked with Ki = approximately 40 microM, and the HERG-selective blocker, E-4031, blocked with Ki = 37 nM. Implications of the presence of HERG-like K+ channels for the ontogeny of microglia are discussed.

Regulation of native Kv1.3 channels by cAMP-dependent protein phosphorylation.

We present evidence that activity of native Kv1.3 channels in human T lymphocytes can be increased by inhibiting phosphatases [using okadaic acid (OA)] or by activating protein kinase A (PKA). OA increased the maximal conductance (Gmax) by 40% and shifted the voltage dependence of activation and inactivation, resulting in a significant increase in window current around the normal membrane potential. PKA inhibition [using the PKA inhibitor peptide PKI-(5-24)] decreased Gmax by 43%, whereas PKA activation [by the Sp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Sp-cAMPS)] increased Gmax by 60% and shifted the inactivation curve, producing an increase in the window current. These results are consistent with our previously published work using cell-attached patches but differ from some studies of Kv1.3. Because we previously reported a similar upregulation by protein kinase C (PKC) activation in these cells, we tested whether the PKA and PKC effects were additive. Our results suggest that PKC-dependent phosphorylation acts as a master switch, inasmuch as calphostin C greatly inhibited the current even after Sp-cAMPS, OA, or PKC activation was used to increase protein phosphorylation. Inasmuch as phosphorylation by both kinases (phorbol ester followed by Sp-cAMPS) abrogated the effects of either kinase alone, our results support the view that Kv1.3 is regulated in a complex manner by serine/threonine phosphorylation.

Native Kv1.3 channels are upregulated by protein kinase C.

The voltage-gated potassium channel, Kv1.3, which is highly expressed in a number of immune cells, contains concensus sites for phosphorylation by protein kinase C (PKC). In lymphocytes, this channel is involved in proliferation-through effects on membrane potential, Ca2+ signalling, and interleukin-2 secretion-and in cytotoxic killing and volume regulation. Because PKC activation (as well as increased intracellular Ca2+) is required for T-cell proliferation, we have studied the regulation of Kv1.3 current by PKC in normal (nontransformed) human T lymphocytes. Adding intracellular ATP to support phosphorylation, shifted the voltage dependence of activation by +8 mV and inactivation by +17 mV, resulting in a 230% increase in the window current. Inhibiting ATP production and action with "death brew" (2-deoxyglucose, adenylylimidodiphosphate, carbonyl cyanide-m-chlorophenyl hydrazone) reduced the K+ conductance (GK) by 41 +/- 2%. PKC activation by 4 beta-phorbol 12,13-dibutyrate, increased GK by 69 +/- 6%, and caused a positive shift in activation (+9 mV) and inactivation (+9 mV), which resulted in a 270% increase in window current. Conversely, several PKC inhibitors reduced the current. Diffusion into the cell of inhibitory pseudosubstrate or substrate peptides reduced GK by 43 +/- 5% and 38 +/- 8%, respectively. The specific PKC inhibitor, calphostin C, potently inhibited Kv1.3 current in a dose- and light-dependent manner (IC50 approximately 250 nM). We conclude that phosphorylation by PKC upregulates Kv1.3 channel activity in human lymphocytes and, as a result of shifts in voltage dependence, this enhancement is especially prevalent at physiologically relevant membrane potentials. This increased Kv1.3 current may help maintain a negative membrane potential and a high driving force for Ca2+ entry in the presence of activating stimuli.

Properties of K+ and Cl- channels and their involvement in proliferation of rat microglial cells.

Essentially pure (>95%) cultures of microglia were established from neopallia of newborn rats and used for whole-cell patch-clamp recording of electrophysiological properties and for proliferation studies. Two types of cultures were examined: 1) "Primary" cultures were grown in culture medium with serum and used within 3 weeks of isolation; 2) and "Colony-stimulating factor (CSF)-1-stimulated" cultures were derived from 3-week-old "primary" cultures by passaging and culturing them for several weeks longer in the presence of conditioned medium enriched in CSF-1. Microglia in the "primary" cultures expressed: 1) an inwardly rectifying K+ current (Kir) that was inhibited by Ba2+; 2) an outwardly rectifying K+ current (Kv) with many similarities to the cloned Kv1.3 channel of lymphocytes, including block by nanomolar concentrations of charybdotoxin (ChTX) and margatoxin (MgTX); and 3) an outwardly rectifying anion current with time- and voltage-independent gating. The anion current is activated reversibly under cell swelling conditions, i.e., after exposure to a hypo-osmotic bathing medium. The anion channels are highly permeable to Cl-, measurably permeable to gluconate (P(gluconate)/ PCl = 0.34), and blocked by flufenamic acid, 4-nitro-2-(3-phenylpropylamino)- benzoic acid (NPPB), and 6, 7-dichloro-2-cyclopentyl-2, 3-dihydro-2-methyl-1-oxo-1H-inden-5-yl (oxy) acetic acid (IAA-94). Microglia in the "CSF-1-stimulated" cultures expressed Kir and Cl- current, but not Kv current. Proliferation in the latter type of cultures could be slowed by omission of the CSF-1 enriched supernatant for 2 days and stimulated by adding back the conditioned medium. This "CSF-1-stimulated" proliferation was inhibited by Ba2+ (Kir blocker), and the Cl(-)-channel blockers flufenamic acid, NPPB, and IAA-94, whereas the Kv blockers ChTX and MgTX had no effect. Thus, Kir and Cl- channels appear to be necessary for "CSF-1-stimulated" proliferation of rat microglia, and there is no evidence that even a transient activation of Kv is necessary.

Chloride-channel block inhibits T lymphocyte activation and signalling.

Both large- and small-conductance chloride (Cl-) channels have been found in human T lymphocytes; however, apart from possible roles in mediating regulatory volume decrease, their functions are not understood. We have used patch-clamp electrophysiology, Ca2+ spectrofluorometry, and Western blot assay for phosphotyrosine to investigate the effects of blocking Cl- channels on proliferation and on specific events in the activation of normal human T cells. Four chemically distinct Cl- channel blockers inhibited both the small-conductance Cl- channels and phytohemagglutinin (PHA)-induced lymphocyte proliferation in a similar dose-dependent manner; their order of potency was 5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB) > 4,4'-diisothiocyano-2,2'-disulfonic acid (DIDS) > flufenamic acid >> IAA-94. The Cl- channel blockers inhibited both the PHA-induced mobilization of Ca2+ and the rapid tyrosine phosphorylation of several polypeptides. Cell proliferation was not rescued by the Ca+ ionophore ionomycin or by addition of exogenous interleukin-2 (IL-2). Moreover, the blockers also inhibited phosphotyrosine expression in IL-2-treated, activated lymphoblasts. Thus, our results support a role for Cl- channels in early, PHA-evoked signalling and in later, II-2-dependent stages of lymphocyte activation and proliferation.

Small-conductance chloride channels in human peripheral T lymphocytes.

During whole-cell patch-clamp recording from normal (nontransformed) human T lymphocytes a chloride current spontaneously activated in > 98% of cells (n > 200) in the absence of applied osmotic or pressure gradients. However, some volume sensitivity was observed, as negative pressure pulses reduced the current. With iso-osmotic bath and pipette solutions the peak amplitude built up (time constant approximately 23 sec at room temperature), a variable-duration plateau phase followed, then the current ran down spontaneously (time constant approximately 280 sec). The anion permeability sequence, calculated from reversal potentials was I-, Br- > NO3-, Cl- > CH3SO3-, HCO3- > CH3COO- > F- > aspartate, gluconate, SO4(2-) and there was no measurable monovalent cation permeability. The Cl- current was independent of time during long voltage steps and there was no evidence of voltage-dependent gating; however, the current showed intrinsic outward rectification in symmetrical Cl- solutions. The conductance of the channels underlying the whole-cell current was calculated from fluctuation analysis, using power-spectral density and variance-vs.-mean analysis. Both methods yielded a single channel conductance of about 0.6 pS at -70 mV (close to the normal resting potential of T lymphocytes). The power spectral density function was best fit by the sum of two Lorentzian functions, with corner frequencies of 30 and 295 Hz, corresponding to mean open times of 0.54 and 5.13 msec. The pharmacological profile included rapid block by external application of flufenamic acid (50 microM), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 100 microM), [6,7-dichloro-2-cyclopentyl-2,3- dihydro-2-methyl-1-oxo-1H-inden-5-yl)oxy] acetic acid (IAA-94, 250 microM) or 100 microM 1,9-dideoxyforskolin. The stilbene derivatives DIDS (4,4'-diisothiocyano-2,2' disulphonic acid stilbene, 500 microM) and SITS (4-acetamido-4'-isothiocyano-2,2'-disulphonic acid stilbene, 500 microM) prevented buildup of Cl- current after a 30-min preincubation at 500 microM. When tested in a mitogenic assay, DIDS, flufenamic acid, NPPB and IAA-94 all inhibited T-cell proliferation, suggesting a physiological function in addition to the observed volume sensitivity.

Intracellular Ca2+ signaling induced by osmotic shock in human T lymphocytes.

After exposure to hypo-osmotic medium most vertebrate cells are able to reregulate their volume by losing electrolytes (K, Cl) and water, a process called regulatory volume decrease (RVD). The whole process of RVD requires a sensor(s) to detect swelling, a transducer(s) to translate the signal, and effectors that cause electrolyte loss. In many cell types an increase in cytoplasmic calcium (Cai) is the transducer and T lymphocytes were formerly thought to fit this pattern. However, this model was thrown into doubt by experiments on Cai-depleted T cells and by the previous failure to detect Cai changes. In the present study we used Ca fluorescence measurements of fluo-3-loaded normal human T lymphocytes exposed to 60% hypo-osmotic saline in a perfused cuvette. We show that hypo-osmotic shock causes a rapid rise in Cai (averaged over approximately 10(4) cells) due to both release of Ca from internal stores and influx. Ca2+ influx occurred at room temperature as well as at 37 degrees C and at a variety of external Ca2+ concentrations (1, 1.5, 2.5 mM). Following hypo-osmotic shock, reexposure to normal osmolarity restored Cai to resting levels. Cell viability and biological responsiveness were not impaired by these osmotic treatments and the subsequent biphasic Cai rise in response to a mitogenic lectin was normal. Using the whole-cell, patch-clamp technique we have isolated an inward cation current that can be carried by Ca2+. Both this current and the Cai rise were blocked by micromolar gadolinium; hence, this current may provide the Ca2+ influx pathway during a hypo-osmotic shock. Finally, these results and recent information on K, Cl, and cation channels in human T cells are incorporated into a model for RVD in these cells.

Criteria for perforated-patch recordings: ion currents versus dye permeation in human T lymphocytes.

Nystatin-perforated patches are now frequently used to help preserve cytoplasmic integrity during patch-clamp recordings. We used voltage-dependent K+ currents in human T lymphocytes to compare conventional whole-cell recordings with perforated-patch recordings (PPR). Although there were pronounced differences in the inactivation kinetics, we discovered that our PPR recordings were not "intact". In every case, coinciding with the gradual capacitance transient increase and decrease in access resistance, we observed that large (> MW 800) fluorescent dyes enter the cell from the pipette. These results suggest that caution is required when using differences in the properties of the currents to confirm that the nystatin-containing patch is intact.