ABSTRACT

Rapid inactivation of Ca 2+ release-activated Ca 2+ (CRAG) channels was studied in Jurkat leukemic T lymphocytes using whole-ceU patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca 2 § the Ca z+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 inV. Several lines of evidence suggest that the fast inactivation process is Ca 2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca 2+ entry through open channels. Second, inactivation is substantially reduced when Ba 2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 raM), a rapid Ca 2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 raM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i . These results demonstrate that rapid inactivation of lc~c results from the action of Ca z+ in close proximity to the intracellular mouths of individual channels, and that Ca 2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca z+ diffusion in the presence of a mobile buffer predicts multiple Ca z+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.

Increased intracellular

Feedback regulation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole cell recording and [Ca2+]i measurement techniques. CRAC channels were activated by passively depleting intracellular Ca2+ stores in the absence of extracellular Ca2+. Under conditions of moderate intracellular Ca2+ buffering, elevating [Ca2+]o to 22 mM initiated an inward current through CRAC channels that declined slowly with a half-time of approximately 30 s. This slow inactivation was evoked by a rise in [Ca2+]i, as it was effectively suppressed by an elevated level of EFTA in the recording pipette that prevented increases in [Ca2+]i. Blockade of Ca2+ uptake into stores by thapsigargin with or without intracellular inositol 1,4,5-trisphosphate reduced the extent of slow inactivation by approximately 50%, indicating that store refilling normally contributes significantly to this process. The store-independent (thapsigargin-insensitive) portion of slow inactivation was largely prevented by the protein phosphatase inhibitor, okadaic acid, and by a structurally related compound, 1-norokadaone, but not by calyculin A nor by cyclosporin A and FK506 at concentrations that fully inhibit calcineurin (protein phosphatase 2B) in T cells. These results argue against the involvement of protein phosphatases 1, 2A, 2B, or 3 in store-independent inactivation. We conclude that calcium acts through at least two slow negative feedback pathways to inhibit CRAC channels. Slow feedback inhibition of CRAC current is likely to play important roles in controlling the duration and dynamic behavior of receptor-generated Ca2+ signals.

One mechanism cytotoxic T lymphocytes use to kill targets is exocytosis of cytotoxic agents from lytic granules, a process that requires Ca(2+) influx. We investigated the role of Ca(2+) influx in granule exocytosis using TALL-104 human leukemic cytotoxic T cells triggered via a bispecific antibody containing an anti-CD3 F(ab') to kill Raji B lymphoma cells. Using a novel fluorescence method, we detected target-directed release of approximately 15% of lytic granules during killing. Consistent with previous work, we observed sustained CTL Ca(2+) gradients during killing, but gradients reflect the behavior of Fura-2 in granules. Rapid imaging experiments suggest that Ca(2+) channels are not polarized during killing, indicating that Ca(2+) influx does not direct granule reorientation. Furthermore, we find that Ca(2+) acts via a high-affinity interaction to promote granule exocytosis.