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L C Schlichter

Publications and source records attributed to L C Schlichter.

35 records · Page 2Linked to original sources
PubMed1992

Cl- channels in intact human T lymphocytes.

We recently described a large, multiple-conductance Cl- channel in excised patches from normal T lymphocytes. The properties of this channel in excised patches are similar to maxi-Cl- channels found in a number of cell types. The voltage dependence in excised patches permitted opening only at nonphysiological voltages, and channel activity was rarely seen in cell-attached patches. In the present study, we show that Cl- channels can be activated in intact cells at physiological temperatures and voltages and that channel properties change after patch excision. Maxi-Cl- channels were reversibly activated in 69% of cell-attached patches when the temperature was above 32 degrees C, whereas fewer than 2% of patches showed activity at room temperature. Upon excision, the same patches displayed large, multiple-conductance Cl- channels with characteristics like those we previously reported for excised patches. After patch excision, warm temperatures were not essential to allow channel activity; 37% (114/308) of inside-out patches had active channels at room temperature. The voltage dependence of the channels was markedly different in cell-attached recordings compared with excised patches. In cell-attached patches, Cl- channels could be open at cell resting potentials in the normal range. Channel activation was not related to changes in intracellular Ca2+ since neither ionomycin nor mitogens activated the channels in cell-attached patches, Ca2+ did not rise in response to warming and the Cl- channel was independent of Ca2+ in inside-out patches. Single-channel currents were blocked by internal or external Zn2+ (100-200 microM), 4-acetamido-4' isothiocyanostilbene-2,2'-disulfonate (SITS, 100-500 microM) and 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS, 100 microM). NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) reversibly blocked the channels in inside-out patches.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

A perfused-cuvette method for fluorimetric studies of non-adherent cells.

We have developed a system for immobilizing non-adherent cells for use in macroscopic fluorescence measurements in a perfused cuvette. Using normal human T lymphocytes loaded with the fluorescent Ca(2+)-indicator, fluo-3, we have validated the properties of these cells immobilized on clear, non-fluorescent fluorohalocarbon film (Aclar) using the non-charged cell adhesive, Cell-Tak.

Cell Separation↗
PubMed1992

Modulation of potassium channels in intact human T lymphocytes.

1. A voltage-dependent K+ channel called the 'n' type (for 'normal') is the most prevalent ion channel found in whole-cell recordings from T lymphocytes. In whole-cell patch-clamp recordings activity of the n-type channel is affected by mitogenic agents, pH, Ca2+ and temperature but not by cyclic nucleotides. Because channel properties and regulation can depend on cytoplasmic components we sought to reassess the properties of K+ channels in intact, normal human T lymphocytes using cell-attached, patch-clamp recordings. In the present study, we show that the predominant K+ channel in resting, intact cells is the n type and is affected by voltage, temperature and Ca2+ in ways similar to the disrupted cell. Moreover, K+ channels are activated by agents that raise cyclic AMP in intact cells. 2. In cell-attached recordings, we found voltage-activated K+ channels in about 60% of patches at room temperature. The channel was K+ selective as judged from the reversal potential under different Ka(+)-K+ gradients and at different resting membrane potentials. Some patches were subsequently excised and the selectivity further confirmed. The current-voltage relation was inwardly rectifying under symmetrical K+ concentrations and had a slope conductance of 9.4 pS at 50 mV depolarized and 23.8 pS at 50 mV hyperpolarized from the resting potential. From the reversal potentials under various conditions the cell resting potential was -51 +/- 1 mV in normal NaCl saline and about 0 mV when the bath contained 150 mM-KCl saline. Two other types of K+ channel were seen in resting, intact cells, but were much less common (less than 5% and 11% of patches). A large-conductance K+ channel was seen in less than 1% of inside-out patches. 3. The predominant K+ channel in intact, resting T lymphocytes was confirmed as the n type underlying the whole-cell K+ current evoked by voltage steps. In cell-attached patches there was a low, steady-state level of activity at the resting potential but activity was greatly increased by depolarizing voltage jumps. Steady-state inactivation could be removed by a hyperpolarizing pre-pulse. Ensemble currents constructed by summing channel openings during repeated voltage jumps showed sigmoid kinetics of current activation and a monoexponential decay phase. These kinetics were well fitted by a Hodgkin-Huxley-type n4j kinetic model with time constants very similar to the whole-cell current of disrupted cells. Moreover, the kinetics depended on the external K+ concentration as previous research has shown.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1992

Nonselective cation channels in intact human T lymphocytes.

Nonselective cation channels were found in single channel recordings from cell-attached patches on human T lymphocytes. These channels were active under conditions that should lead to cell swelling (hypotonic bath solutions with NaCl or KCl); however, a definite dependence of activity on cell swelling has not been proven. Under these conditions similar channels were found in 20 of 23 patches from 11 different blood donors. The current-voltage relation was approximately linear for outward current (11-14 pS) and inwardly rectifying (to 23 pS) when the intact cells were depolarized with high KCl in the bath. The voltage dependence of channel activity is consistent with closing at hyperpolarized membrane potentials (Vm less than or equal to -50 mV) and block of open channels at strongly depolarized membrane potentials (Vm greater than 0 mV). Reversal potentials under all ionic gradients tested are consistent with a channel that is poorly selective between Na+ and K+ ions. Active channels in cell-attached patches were rapidly blocked by bath addition of the membrane-permeant inhibitor quinine. Channels that were active in cell-attached became quiescent after patch excision; however, two patches remained active long enough to obtain current-voltage relations. These were linear with a slope conductance for outward current of 8-11 pS. Because of the clustering of single-channel openings, detailed voltage dependence of kinetics and probability of opening were not studied.

Cations↗
PubMed1992

Acute exposure to human interferon-alpha affects ion currents in human natural killer cells.

Interferon-alpha (IFN-alpha) is a particularly potent stimulator of human natural killer (NK) cell activity. The initial trigger for IFN action is not known, but there is indirect evidence from a number of cell types that changes in ion channel activity are among the earliest responses. Previous evidence includes changes in Ca2+ fluxes and intracellular activity, membrane potential changes, and effects of ion-channel blockers. Killing by human NK cells is dependent on external Ca2+ and on K+ channel activity. In the present study we have confirmed this dependence and the augmentation by human IFN-alpha. Then we directly studied the effects of IFN-alpha on ion currents in human NK cells using the patch-clamp electrophysiological techniques. We find that IFN-alpha can increase the predominant K+ current near the resting potential but suppresses it at higher voltages. Within 1 min after acute IFN-alpha treatment a new current is induced. This small current appears to be through nonselective cation channels that allow monovalent and divalent cations, including Ca2+ to permeate. This current presents a possible early triggering mechanism whereby acute exposure to IFN-alpha augments NK cytotoxicity.

Cations, Divalent↗
PubMed1992

Dual action of 2,3-butanedione monoxime (BDM) on K+ current in human T lymphocytes.

Originally developed as antidotes to organophosphorus nerve poisons, the oximes have attracted renewed interest in studies of cellular regulation. In particular, 2,3-butanedione monoxime (BDM) has gained attention as a useful membrane-permeant "chemical phosphatase" for studying roles of protein phosphorylation. It has been proposed that effects of BDM on cardiac muscle tension, action potentials, neuromuscular transmission and ion currents are related to dephosphorylation of substrates as diverse as myofibrils and ion channels. In the present study, voltage-dependent K+ currents in human T lymphocytes were studied using the whole cell patch clamp technique. Preincubating intact cells briefly in 5 mM BDM before recording reduced the K+ current in an irreversible manner, consistent with chemical (phosphatase?) modification of the channels. In contrast, acute BDM treatment produced a rapid, reversible block of K+ current with half block at about 5 mM. Moreover, including adenosine-O-5'-(3-thiotriphosphate) (500 microM) in the patch pipette did not prevent the rapid, reversible block by BDM. Under these conditions, the most likely mechanism was a direct block of channels from the outside. Because similar K+ currents are present in many tissue and cell types, a direct channel block suggests caution in interpreting the effects of oximes as resulting from protein dephosphorylation.

Cholinesterase Reactivators↗
PubMed1990

A large, multiple-conductance chloride channel in normal human T lymphocytes.

Chloride (Cl) channels have been proposed to play roles in lymphocyte functions including volume regulation and cellular cytotoxicity; however, direct studies of such channels in normal human lymphocytes are lacking. In the present study we describe a large conductance Cl channel observed in about 50% of excised, inside-out patches from normal human peripheral T lymphocytes. The channel has multiple conductance states with linear single-channel current-versus-voltage relationships in symmetrical Cl solutions. The most prevalent state is the largest, which has a conductance of about 365 pS. The channel closes in a voltage-dependent manner at both negative and positive potentials, but does not show voltage-dependent inactivation. The probability of opening is maximal between -15 mV and +15 mV and the voltage dependence is well described by two Boltzmann equations with half-maximal probabilities at -22.8 mV and +18.0 mV. The slopes of the voltage dependence suggest two gates in series with 5.7 and 9.6 equivalent charges. The channel was about 30 times more selective for Cl- than for Na+ or K+ under balanced osmolarity but less selective (approx. 11:1) under a large osmotic gradient. The single-channel conductance increased with Cl concentration with an apparent saturation at about 581 pS and a Michaelis-Menten constant of about 120 mM. The selectivity sequence among anions, determined from changes in reversal potential was: I- greater than NO3- greater than Br-, Cl- greater than F-, isethionate, HCO3- greater than SO4(2-) greater than gluconate, propionate greater than aspartate much greater than Na+, K+ and was apparently the same for subconductance states.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1990

Modulation of potassium channels in human T lymphocytes: effects of temperature.

1. The predominant channels found in lymphocytes with patch-clamp whole-cell recordings are voltage-gated K+ channels. Several lines of evidence suggest that these channels are involved in lymphocyte function. Most lymphocyte functions are temperature sensitive and have not been correlated with electrophysiology at different temperatures. We have examined the effect of temperature on the voltage-dependent K+ channel in normal human T lymphocytes. Both macroscopic current and single-channel events were studied with whole-cell recordings at temperatures from 5 to 42 degrees C. 2. Peak conductance, activation rate, inactivation rate and rate of recovery from inactivation all increased progressively as the temperature increased. The effect of temperature on channel opening processes was greater at low temperatures. In contrast, the inactivation process was most sensitive to temperature changes above room temperature. Arrhenius plots of conductance and kinetic parameters were curvilinear with no obvious break-points. 3. The increase in whole-cell conductance at 37 degrees C was due to both an increase in the single-channel conductance and in the probability that each channel is open at any time. 4. K+ currents were fitted by Hodgkin-Huxley equations with n4j kinetics providing the best description of the currents at all temperatures tested. 5. Steady-state activation- and inactivation-voltage curves shifted in opposite directions with warming, resulting in a greater area of overlap of the curves ('window' current). The increase in resting K+ channel activity predicted by a greater window current was confirmed with single-channel measurements. 6. The present study has shown that the behaviour of K+ channels in human T lymphocytes is temperature dependent.

Action Potentials↗
PubMed1989

Ionic currents underlying the action potential of Rana pipiens oocytes.

Ionic currents in immature, ovulated Rana pipiens oocytes (metaphase I) were studied using the voltage-clamp technique. At this stage of maturity the oocyte can produce action potentials in response to depolarizing current or as an "off response" to hyperpolarizing current. Reducing external Na+ to 1/10 normal (choline substituted) eliminated the action potentials and both the negative-slope region and zero-crossing of the I-V relation. Reducing external Cl- to 1/10 or 1/100 normal (methanesulfonate substituted) lengthened the action potential. The outward current was reduced and a net inward current was revealed. By changing external Na+, Cl-, and K+ concentrations and using blocking agents (SITS, TEA), three voltage- and time-dependent currents were identified, INa, IK and ICl. The Na+ current activated at about 0 mV and reversed at very positive values which decreased during maturation. Inward Na+ current produced the upstroke of the action potential. During each voltage-clamp step the Na+ current activated slowly (seconds) and did not inactivate within many minutes. The Na+ current was not blocked by TTX at micromolar concentrations. The K+ current was present only in the youngest oocytes. Because IK was superimposed on a large leakage current, it appeared to reverse at the resting potential. When leakage currents were subtracted, the reversal potential for IK was more negative than -110 mV in Ringer's solution. IK was outwardly rectifying and strongly activated above -50 mV. The outward K+ current produced an after hyperpolarization at the end of each action potential. IK was blocked completely and reversibly by 20 mM external TEA. The Cl- current activated at about +10 mV and was outwardly rectifying. ICl was blocked completely and reversibly by 400 microM SITS added to the bathing medium. This current helped repolarize the membrane following an action potential in the youngest oocytes and was the only repolarizing current in more mature oocytes that had lost IK. The total leakage current had an apparently linear I-V relation and was separated into two components: a Na+ current (IN) and a smaller component carried by as yet unidentified ions.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1989

Interactive effects of Na and K in killing by human natural killer cells.

Contact-mediated lysis by human natural killer cells is inhibited by a number of drugs that block the predominant K channel. In this study we have further examined the role of the K channel and the interactions between passive K and Na transport in killing. Low external Na-inhibited killing and inhibition were not due to reduce inward current through the Na channels in the target cell. A role for the Na/H antiport is suggested since amiloride inhibited killing in a dose-dependent manner that was competitive with external Na. Depolarizing the killer cell with elevated external K did not inhibit killing. On the contrary, high K0 reduced the inhibition caused by low Na0 and by the K-channel blockers quinidine, verapamil, and retinoic acid. Hyperpolarizing the killer cell with low K0 or valinomycin inhibited killing. Valinomycin, which should prevent the depolarization caused by K-channel block, did not reverse the effect of the blockers quinidine, verapamil, and 4-aminopyridine. Hence, the primary role of the K channels during killing is not maintain the negative membrane potential. On the contrary, depolarization may promote killing under conditions where killing is submaximal.

Amiloride↗
PubMed1989

Ca2(+)-activated K+ channels in human B lymphocytes and rat thymocytes.

1. Previous evidence for the existence of Ca2(+)-activated K+ channels in lymphocytes comes from measurements using voltage-sensitive dyes and from tracer flux studies. We have now directly measured these channels in human tonsillar B lymphocytes and rat thymocytes in single-channel recordings from cell-attached and excised patches. 2. In cell-attached recordings, intracellular Ca2+ was raised by either ionomycin or replacement of external Ca2+ following incubation in Ca2(+)-free medium. Indo-1 measurements during the Ca2(+)-replacement technique showed that [Ca2+]i rose from approximately 90 to 260 nM. Both techniques activated two channels of approximately 25 and 8 pS (slope conductance at 0 mV applied, with 140 mM-K+ in the pipette). Over 90% of patches displayed this activity, indicating a high density of these channels in the membrane. 3. Both channels reversed near the K+ equilibrium potential with either KCl or potassium aspartate in the pipette, when the cells were bathed in normal or high-K+ saline. Therefore, these channels are selective for K+. 4. The larger channel was studied in more detail. It displayed inward rectification in symmetrical K+ solutions. The open-channel probability was weakly dependent on membrane potential. 5. Ca2(+)-dependent K+ channels were also recorded from excised, inside-out membrane patches. The threshold for activation was 200-300 nM [Ca2+i]. 6. Patch excision altered some characteristics of IK(Ca). Channels were activated in fewer than 50% of patches and the main conductance level was approximately 34 pS (at -80 mV). The duration of single-channel events was shorter than in cell-attached patches; kinetic analysis suggested that this was due to the loss of an open state in excised patches. 7. We conclude that B and T lymphocytes have K(+)-selective channels which are activated by internal [Ca2+] in the physiological range and which will influence the membrane potential during cell activation.

Action Potentials↗
PubMed1986

Potassium channels in human NK cells are involved in discrete stages of the killing process.

Using the whole-cell variation of the patch-clamp technique, we have found a voltage-dependent K+ current in human natural killer (NK) cells. This K+ current is reduced in a dose-dependent manner by a variety of ion-channel blockers (verapamil, quinidine, 4-aminopyridine, Cd2+) at concentrations comparable to those that inhibit natural killing. Pretreatment of target cells with quinidine or verapamil did not significantly reduce their sensitivity to killing, whereas substantial inhibition of killing was observed after pretreatment of effector cells. Both verapamil and quinidine reduced the proportion of effector-target cell conjugates, suggesting that K channels play a role in the "binding" phase of the killing process. By adding EDTA or channel blockers as various times in a Ca-pulse assay system, we have also delineated a blocker-sensitive phase of bound conjugates that strictly corresponds with the Ca-dependent "programming" stage of killing. In contrast, the killer cell-independent stage, which is Ca2+ independent, apparently does not require functioning K channels. Verapamil and quinidine do not affect target cell sensitivity to the putative soluble mediator of natural killing, natural killer cytotoxic factor (NKCF), but inhibit release of NKCF from NK cells. Thus, the data suggest that K channels in NK cells play essential roles in the natural killing process that include events in the "programming-for-lysis" phase leading to release of NKCF.

4-Aminopyridine↗
PubMed1985

Fertilization-induced ionic conductances in eggs of the frog, Rana pipiens.

Fertilization of the frog egg (Rana pipiens) elicits a positive-going shift in membrane potential (fertilization potential) that lasts 10-20 min and functions as a fast block to polyspermy. We examined the ion conductances underlying the fertilization potential, using the voltage-clamp technique. We measured the membrane capacitance during the fertilization potential by applying an alternating current. We also determined the intracellular K and Cl concentrations in the egg, using ion-selective micro-electrodes. The conductance is largest in the first 2 min after fertilization. Regardless of whether the stimulus is provided by one or by more than one sperm or by artificial activation, the size of the conductance increase is the same, reaching a maximum of about 40 microseconds. Two separate conductances are involved at fertilization: Cl and K. [K]i = 121 mM and [Cl]i = 44 mM. The natural external medium is pond water (approximated in our experiments by 10% Ringer solution); therefore, an increase in K and Cl conductances leads to an efflux of both ions. The equilibrium potential of the fertilization current is between the Cl and K equilibrium potentials (ECl and EK), closer to ECl. 10 mM-external tetraethylammonium (TEA) brings the equilibrium potential close to ECl and reduces the maximum conductance by about half. The Cl conductance is not blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS). The time courses of the K and Cl conductances are similar. The TEA-resistant conductance (primarily Cl conductance) activated at fertilization increases as the membrane potential becomes more positive. A voltage-sensitive Na conductance present in the unfertilized egg disappears after fertilization. During fertilization this conductance is too small to contribute significantly to the fertilization potential. The membrane capacitance increases by an average of 1.9 times in the first 2 min following the rise of the fertilization potential, during the period of cortical vesicle exocytosis. Capacitance then gradually decreases; at 1 h after fertilization, capacitance is 82% of the value in the unfertilized egg. The conductance increase precedes the capacitance increase by several seconds. Therefore the initial appearance of Cl and K channels cannot be accounted for by addition of membrane by cortical vesicle exocytosis. The conductance subsequently decreases, suggesting that the disappearance of the Cl and K channels is not caused by membrane removal.

Action Potentials↗
PubMed1983

Spontaneous action potentials produced by Na and Cl channels in maturing Rana pipiens oocytes.

The electrical excitability of maturing Rana pipiens oocytes was studied using intracellular recording and voltage-clamp techniques. Naturally ovulated oocytes, removed from the body cavity within a few hours after ovulation, possess voltage-sensitive Na and Cl channels that can produce action potentials (ap's). Young oocytes (sometime during metaphase I to first polar body stage) can generate trains of spontaneous action potentials: no chemical treatment or current injection is required. This is the first report of spontaneous repetitive firing in an egg cell membrane. As the oocyte matures, the duration of each ap increases because the outward Cl- current decreases. Middle-aged oocytes (about first polar body stage to metaphase II) have continuously positive membrane potentials (Vm's). Mature, activatable (metaphase II) oocytes have negative Vm's when impaled but can produce a long-lived ap when depolarizing current is injected. The ap's differ fundamentally from ap's in other excitable cells, including eggs: the Na+ current develops slowly and does not inactivate; most of the outward current is carried by Cl-, not by K+; the Cl channel is lost or is rendered insensitive to voltage as the oocyte matures.

Action Potentials↗
PubMed1983

A role for action potentials in maturing Rana pipiens oocytes.

Intracellular electrical recording and voltage-clamp techniques were used to investigate possible roles for the action potentials (ap's) of Rana pipiens oocytes. The peak of each spontaneous or evoked ap is at or near the equilibrium potential for Na; therefore, the internal Na+ activity (aiNa) can be calculated from the Nernst equation. During maturation from metaphase I to metaphase II, aiNa increases from about 5 to 23 mM. By using three methods to prevent ap's from firing (voltage clamping, increasing the mechanical damage, adding 5 mM CoCl2 to the Ringer's) it was shown that the increase in aiNa required the existence of ap's. External Co2+ appeared to prevent the ap's by blocking only the Na+ current. Calculations showed that the Na+ influx during an ap could account for the observed increase in aiNa if a gradient or nonuniform distribution of Na+ exists within the oocyte. Preventing the ap's also delayed the onset of shock activatibility, a criterion of maturity. I propose that ap's load the oocyte with Na+ which may regulate the rate of maturation either directly or indirectly.

Action Potentials↗