Lyanne Schlichter

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− > NO3−> Br−, Cl− > F−, isethionate, HCO3−> SO42−> gluconate, propionate > aspartate ≫ Na+, K+ and was apparently the same for subconductance states. The sequence determined from measurable values of single-channel conductance was: I− > NO3−> Br− > Cl− > F− > HCO3−, isethionate. The channel was rapidly and reversibly blocked by 1 mM Zn2+ or 1 mM Ni2+ added to the cytoplasmic face. Possible roles of this maxi-Cl channel in lymphocyte function are discussed.

Cl− channels in intact human T lymphocytes

SummaryWe 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 maxiCl− 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 cellattached patches when the temperature was above 32°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. Singlechannel currents were blocked by internal or external Zn2+ (100–200 μm), 4-acetamido-4′ isothiocyanostilbene-2,2′-disulfonate (SITS, 100–500 μm) and 4,4′-diisothiocyanostilbene 2,2′disulfonate (DIDS, 100 μm). NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) reversibly blocked the channels in inside-out patches.

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)

Uptake properties of zidovudine (ZDV) by an HIV‐I target brain parenchyma cell line

Clinical Pharmacology & Therapeutics (1999) 65, 125–125; doi:

Transport properties of thymidine by a rat microglia cell line

Clinical Pharmacology & Therapeutics (1999) 65, 124–124; doi: