VOLTAGE-GATED AND CA2+-ACTIVATED K+ CHANNELS IN INTACT HUMAN T-LYMPHOCYTES - NONINVASIVE MEASUREMENTS OF MEMBRANE CURRENTS, MEMBRANE-POTENTIAL, AND INTRACELLULAR CALCIUM

Voltage-gated n-type K(V) and Ca2+-activated K+ [K(Ca)] channels were studied in cell-attached patches of activated human T lymphocytes. The single-channel conductance of the K(V) channel near the resting membrane potential (V-m) was 10 pS with low K+ solution in the pipette, and 33 pS with high K+ solution in the pipette. With high K+ pipette solution, the channel shuwed inward rectification at positive potentials. K(V) channels in cell-attached patches of T lymphocytes inactivated more slowly than K(V) channels in the whole-cell configuration. In intact cells, steady state inactivation at the resting membrane potential was incomplete, and the threshold for activation was close to V-m. This indicates that the K(V) channel is active in the physiological V-m range. An accurate, quantitative measure for V-m was obtained from the reversal potential of the K(V) current evoked by ramp stimulation in cell-attached patches, with high K+ solution in the pipette. This method yielded an average resting V-m for activated human T lymphocytes of -59 mV. Fluctuations in V-m were detected from changes in the reversal potential. Ionomycin activates K(Ca) channels and hyperpolarizes V-m to the Nernst potential for K+. Elevating intracellular Ca2+ concentration ([Ca2+](i)) by ionomycin opened a 33-50-pS channel, identified kinetically as the CTX-sensitive IK-type K(Ca) channel. The Ca2+ sensitivity of the K(Ca) channel in intact cells was determined by measuring [Ca2+](i) and the activity of single K(Ca) channels simultaneously. The threshold for activation was between 100 and 200 nM; half-maximal activation occurred at 450 nM. At concentrations >1 mu M, channel activity decreased. Stimulation of the T-cell receptor/CD3 complex using the mitogenic lectin, PHA, increased [Ca2+](i), and increased channel activity and current amplitude resulting from membrane hyperpolarization.