Temperature-dependent Block of Capacitative Ca2+ Influx in the Human Leukemic Cell Line KU-812

The mechanism by which depletion of intracellular Ca 2+ stores activates Ca 2+ influx is not understood. We recently showed that primaquine, an inhibitor of vesicular transport, blocks the activation of the calcium release-activated calcium current ( I CRAC ) in rat megakaryocytes (Somasundaram, B., Norman, J. C., and Mahaut-Smith, M. P. (1995) Biochem. J . 309, 725-729). Since it is well established that vesicular transport is temperature-sensitive, we have investigated the effect of temperature on both the activation and maintenance of store-mediated Ca 2+ and Mn 2+ influx in the human leukemic cell line KU-812 using a combination of whole cell I CRAC recordings and measurements of Mn 2+ photoquench of fura-2. Activation of I CRAC was temperature-sensitive, showing a nonlinear reduction when the temperature was lowered from 27 to 17°C with an abrupt change at 21-22°C and complete inhibition at 17°C. Once activated, I CRAC also displayed an abrupt reduction at 21-22°C but was not completely blocked even when the temperature was reduced to 14°C, suggesting that at least one of the temperature-sensitive components is exclusively involved in I CRAC activation. Activation of store-mediated Mn 2+ influx also showed similar nonlinear temperature sensitivity and complete inhibition at 19°C. However, in contrast to I CRAC measurements, lowering the temperature following maximal activation of the influx pathway at 37°C did not result in any detectable residual Mn 2+ entry below 19°C. We conclude that the mechanism of store-mediated Ca 2+ influx involves temperature-dependent steps in both its maintenance and activation, suggesting dependence on a lipid membrane environment.