Abstract
The mechanism by which depletion of intracellular Ca2+ stores activates Ca2+ influx is not understood. We recently showed that primaquine, an inhibitor of vesicular transport, blocks the activation of the calcium release-activated calcium current (ICRAC) 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 Ca2+ and Mn2+ influx in the human leukemic cell line KU-812 using a combination of whole cell ICRAC recordings and measurements of Mn2+ photoquench of fura-2. Activation of ICRAC was temperature-sensitive, showing a nonlinear reduction when the temperature was lowered from 27 to 17 degrees C with an abrupt change at 21-22 degrees C and complete inhibition at 17 degrees C. Once activated, ICRAC also displayed an abrupt reduction at 21-22 degrees C but was not completely blocked even when the temperature was reduced to 14 degrees C, suggesting that at least one of the temperature-sensitive components is exclusively involved in ICRAC activation. Activation of store-mediated Mn2+ influx also showed similar nonlinear temperature sensitivity and complete inhibition at 19 degrees C. However, in contrast to ICRAC measurements, lowering the temperature following maximal activation of the influx pathway at 37 degrees C did not result in any detectable residual Mn2+ entry below 19 degrees C. We conclude that the mechanism of store-mediated Ca2+ influx involves temperature-dependent steps in both its maintenance and activation, suggesting dependence on a lipid membrane environment.
Highlights
We recently provided evidence to further support this notion by showing that two established inhibitors of vesicle-mediated protein transport, the antimalarial amine primaquine and GTP␥S, block the appearance of ICRAC in response to store depletion in rat megakaryocytes (Somasundaram et al, 1995)
In this study, we have investigated the effect of temperature on the activation and maintenance of storemediated Ca2ϩ influx in a human leukemic cell line, KU-812 (Nakazawa et al, 1989), using a combination of whole cell patch clamp recordings and fluorescent photoquench of fura-2
The present study demonstrates that, in KU-812 cells, storeregulated Ca2ϩ and Mn2ϩ entry is strongly dependent on temperature
Summary
Store depletion-activated currents with different properties from ICRAC have been described in some cell types (Luckhoff and Clapham, 1994; Vaca and Kunze 1994; Vaca et al 1994); ICRAC appears to be the predominant Ca2ϩ influx pathway in nonexcitable cells (Hoth and Penner 1993; Zweifach and Lewis, 1993; Somasundaram and Mahaut-Smith 1994; for review, see Fasalato et al 1994). ICRAC is distinguishable from other store-dependent influx currents by being highly selective for Ca2ϩ and having a single channel conductance below the resolution of patch clamp recordings (Hoth and Penner, 1993; Zweifach and Lewis, 1993). We recently provided evidence to further support this notion by showing that two established inhibitors of vesicle-mediated protein transport, the antimalarial amine primaquine and GTP␥S, block the appearance of ICRAC in response to store depletion in rat megakaryocytes (Somasundaram et al, 1995). The KU-812 cell line was selected because it displayed a high ICRAC channel density and an ability to load and retain the Ca2ϩ-sensitive dye fura-2 and because of the availdiethyl-N,NЈ-[(4R5R)-4,5-dimethyl-1,8-dioxo-3,6-dionachamethylene]bis(12-methylaminododecanoate)
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