MODULATION OF POTASSIUM CHANNELS IN INTACT HUMAN LYMPHOCYTES-T

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 Na+-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 (< 5% and 11% of patches). A large-conductance K+ channel was seen in < 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. Steady-state activity in cell-attached patches showed the same voltage dependence as we previously predicted from activation and inactivation curves of whole-cell currents. Activity was maximal at about -30 mV and the voltage dependence in whole-cell recordings was shifted 10-15 mV in the hyperpolarizing direction. 4. These K+ channels in intact cells were modulated by temperature and internal Ca2+ in much the same way as previously shown for whole-cell and excised-patch recordings. K+ channel activity at the resting potential increased about 10-fold when the temperature was raised from 22 to 30-degrees-C and the single-channel conductance increased with a temperature coefficient (Q10) of about 1.2. This voltage-dependent K+ channel was inhibited by a rise in internal Ca2+ concentration produced by ionomycin in the presence of external Ca2+. In cell-attached patches expressing n-type channel activity, several agents that raise cyclic AMP levels in T lymphocytes caused a dramatic increase in K+ channel activity with single-channel events showing some characteristics of the n-type channel; that is, conductance, reversal potential and activation during depolarizing voltage jumps. However, there was a difference in channel voltage dependence since K+ channel activity was enhanced over a wide voltage range, including the cell resting potential. Hence, these K+ channels may not be the n type. Agents we used to raise cyclic AMP included the analogue 8-bromo-cyclic AMP, the receptor agonists isoprenaline, histamine and prostaglandin E2, the adenylate cyclase activator, forskolin and the phosphodiesterase inhibitor, isobutyl methyl xanthine. In contrast with intact cells, cyclic AMP had no effect in whole-cell recordings. Cyclic AMP does not apparently affect the resting intracellular Ca2+ levels in intact T lymphocytes. Exposure of intact T lymphocytes to up to 500-mu-M-8-bromo-cyclic AMP, dibutyryl cyclic AMP or chlorphenylthio-cyclic AMP produced no change in intracellular Ca2+, as measured by Fluo-3 fluorescence at room temperature. Levels of Ca2+ in batches of cells were 96 +/- 5 nM (n = 5) before and after 100-500-mu-M-dibutyryl cyclic AMP; 86 +/- 7 nM (n = 3) before and after 100-500-mu-M-chlorphenylthio-cyclic AMP and 87 +/- 10 nM (n = 7) before and after 100-500-mu-M-8-bromo-cyclic AMP.