Temperature-dependent block of capacitative Ca2+ influx in the human leukemic cell line KU-812.
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.
Somasundaram
B
B
The Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom.
Mahaut-Smith
M P
MP
Floto
R A
RA
D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007938:Leukemia; D008345:Manganese / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D011319:Primaquine / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D013696:Temperature; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; D005456:Fluorescent Dyes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008345:Manganese; D004533:Egtazic Acid; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D012964:Sodium; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D011319:Primaquine; D011188:Potassium; D002118:Calcium; D016257:Fura-2