Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels.
In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.
Moreau
Ben
B
Laboratory of Cellular and Molecular Signalling, Department of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, United Kingdom.
Straube
Sebastian
S
Fisher
Richard J
RJ
Putney
James W
JW
Parekh
Anant B
AB
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D015220:Calcium Channels / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000502:physiology*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology / Q000502:physiology; D007700:Kinetics; D015471:Leukemia, Basophilic, Acute; D018408:Patch-Clamp Techniques; D051381:Rats; D019284:Thapsigargin / Q000494:pharmacology
D015220:Calcium Channels; D002147:Calmodulin; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium